key: cord- - lmq s l authors: salvadori, marcia r.; yamada, aureo t.; yano, tomomasa title: morphological and intracellular alterations induced by cytotoxin vt y produced by escherichia coli isolated from chickens with swollen head syndrome date: - - journal: fems microbiology letters doi: . /s - ( ) - sha: doc_id: cord_uid: lmq s l abstract recently, a novel verocytotoxin named vt y was described which belongs to the stx family and is produced by escherichia coli isolated from domestic poultry with swollen head syndrome (shs). the vt y toxin induced apoptosis in vero, hela, cho, cef (primary chicken embryo fibroblast) and pck (primary chicken kidney) cell lines. morphological evidence (nuclear shrinkage, chromatin condensation and blebbing of the plasma membrane) of apoptosis could be distinguished in min and was maximal at h after treatment with vt y. this was confirmed by the terminal dutp nick-end-labeling (tunel) method. swollen head syndrome (shs) in avian species was ¢rst described by morley and thomson [ ] who attributed the disease to a mixed infection with escherichia coli and coronavirus, paramyxovirus, or pneumovirus in which the initial viral infection caused acute rhinitis that permitted e. coli to invade the subcutaneous facial tissues [ ] . this acute respiratory disease observed in domestic poultry is characterized by swelling of the peri-and infra-orbital sinuses, torcicollis, opisthotonus and lack of coordination [ , ] . since the syndrome was described in south africa, in , there has been progressive spread to many other countries. verocytotoxin-producing e. coli (vtec) are associated with infant diarrhea, hemorrhagic colitis and hemolyticû remic syndrome in humans, dysentery in calves, edema disease (ed) of pigs and, more recently, shs in birds [ , , , ] . vtec strains produce one or more vts: vt , and several variants of vt . the vt v is produced by e. coli isolated from pigs with ed [ ] . recently par-reira and yano [ ] described a novel cytotoxin called vt y and sequencing of a -bp dna fragment of vt y showed that it contained the stx gene of shigella dysenteriae, but the region bp upstream of the start of the gene for the stxa subunit was identical to the corresponding sequence for e. coli o :h-, except for nucleotide, suggesting that vt y is a member of the stx family [ ] . the aim of this study is to illustrate the morphological and intracellular alterations induced in vitro by cytotoxin vt y in vero, hela, cho, hep- , pck and cef cell lines. . . bacterial strains e. coli shs- strain isolated from chickens with shs was used as a source of vt y [ ] . e. coli strain k c was used as a negative control and strain j (o :h-vt ) was used as a source of vt (kindly donated by yoshifumi takeda, japan) in these experiments. all e. coli strains were stored at ³c in luria^bertani (lb) [ ] broth to which % glycerol was added. the cytotoxin vt y was puri¢ed [ ] , and protein content was assayed as described by bradford [ ] . the cytopathic e¡ect (cpe) of puri¢ed vt y was examined in vero (african green monkey kidney), hela (human epitheloid cervical carcinoma), cho (chinese hamster ovary), hep- (human larynx epidermoid carcinoma), pck (primary chicken kidney) cell lines obtained from the laboratory of cell culture, dmi, unicamp, brazil; cef (chicken embryo ¢broblast) cells were generously supplied by fort dodge laboratories, inc. campinas, brazil. all cell lines were maintained as described by mac-leod and gyles [ ] . the cells were grown at ³c in eagle's minimal essential medium (emem) with earle's salts (cultilab, campinas, sp, brazil), supplemented with % fetal bovine serum (fbs, sigma, st. louis, mo, usa), penicillin ( u ml ) and streptomycin ( wg ml ). to determine cytotoxic activity [ ] , wl of medium and u cells per well were placed in -well plastic plates (costar, cambridge, ma, usa). after h, culture medium was aspirated and wl of fresh emem without fbs, and wl ( ng ml ) of puri¢ed vt y were added to each well. the plates were incubated at ³c in a % co atmosphere for h. morphological changes were observed over the -h period and the test was scored as positive if more than % of the cells exhibited cytotoxic e¡ects. control e. coli strains were j (o :h-vt ) and k c . the cellular viability was determined using a neutral red ( -amino- -methyl- -dimethyl-amino-phenazoniumchloride) assay [ ] . the neutral red assay was performed in vero, hela, cef, pck and cho cells. puri¢ed vt y cytotoxin was applied to the cells and after h, the medium was substituted with . ml of neutral red solution ( wg ml ) per well and the plate incubated at ³c in % co for h, to allow for dye incorporation. afterwards, the dye medium was removed and each well was rapidly washed with formol^calcium solution ( % formaldehyde and % calcium chloride) to remove unincorporated neutral red; . ml of solution containing ml acetic acid with ethanol % was added to each well and the plate kept for min at room temperature to extract the dye from the cells. controls consisted of toxin-treated and untreated cells but without addition of neutral red, (blank). normal cells, without addition of cytotoxin, but with addition of neutral red, were utilized as controls of % with viable cells. the color reaction was measured with an elisa plate reader (multiskan bichromatic) at nm. the cd , was de¢ned as the minimum amount of vt y required to kill % of the cells ( cd ), and was determined from the toxin concentration resulting in % neutral red absorbance compared to control cells as %. leakage of the cytosolic enzyme ldh, was assessed [ ] to investigate whether vt y a¡ected the integrity of the plasma membrane of vero cells. medium from vero cells treated with cd of vt y or without vt y was collected after , , , min and immediately chilled on ice until measurement of ldh activity. a fraction of each sample was added to a cuvette containing sodium pyruvate in ml freshly prepared potassium phosphate bu¡er ( . m, ph . ) and the mixture was equilibrated for min at ³c. the enzyme activity was determined spectrophotometrically and the rate of enzyme leakage min was calculated and expressed as percentage of total ldh activity as compared to untreated control cells. vt y in vero, hela, cef, pck and cho cells the vero, hela, cef, pck and cho cells were grown on coverslips in -well plates using cells ml per well in a volume of ml. the plates were incubated at ³c in % co . after h, culture medium was aspirated and replaced with ml of fresh medium. then, cd vt y was added to each well, and incubated for the same time intervals as in the ldh assay. after incubation, the coverslips were washed with pbs, ¢xed in % formaldehyde solution in pbs for h, then washed with distilled water and stained with . % toluidine blue in mcilvaine ph . bu¡er [ ] . the coverslips were washed for min with distilled water, air-dried, cleared in xylene and mounted using entellan (merck, germany). dna fragmentation in vt y-treated and untreated vero cells was investigated in situ by the terminal dutp nick-end-labeling (tunel) method using an in situ cell death detection pod kit (roche, mannheim, germany). experiments were performed according to the manufacturer's protocol. the vt y was cytotoxic to vero, hela, cef, pck and cho cells, but not for hep- cells. by light microscopy the cytotoxin-a¡ected cells appeared round and shrivelled, and the cells were dead within h. the neutral red assay showed that vero cells were more sensitive than other cells tested since % cell death was observed in the presence of . ng ml vt y. a linear increase in the percentage of dead cells was found as protein concentration was increased within the range indicated in fig. . after min minimal ( %) ldh leakage was observed as compared to the control (fig. ) , but by min an increase to % above control ldh leakage had occurred. the cytotoxin vt y caused morphological alterations in vero cells, such as cytoplasm vacuolization, blebbing of the plasma membrane, chromatin condensation within the nucleus, nuclear shrinkage and apoptotic bodies within min. the e¡ects became more pronounced with time to a¡ect the majority of cells. by h, % cell death was observed (data not shown). when cells were cultured in the presence of vt y, they showed condensed chromatin (fig. b) , cleaved nuclei (fig. c ) and cytoplasmic blebbing (fig. d) , ¢ndings typical of apoptosis. we also observed the presence of cytoplasmic vacuoles in conjunction with apoptotic fea-tures (fig. c) . however, none of these features occurred in control cells (fig. a) . the cleavage of nuclear dna, a major feature of apoptosis, occurred during vt-induced death of vero cells, as con¢rmed by in situ detection by the tunel method (fig. b) . the same vero cells clearly incorporated biotinylated nucleotides, indicating the occurrence of dna fragmentation induced by vt y, in contrast with control cells (fig. a ). programmed cell death, or apoptosis, is de¢ned by the cell's ultrastructural morphology [ ] and is characterized by cell shrinkage, membrane blebbing, and condensation of nuclear chromatin. these morphological changes are accompanied by dna fragmentation, and this form of cell death is a natural process by which an organism removes damaged or unnecessary cells. it may also be triggered by external stimuli [ ] . recently, reports have claimed that vts induce programmed cell death, apoptosis, in cell lines such as vero, mdck [ , ] and others. in our studies, we showed evidence of morphological alterations characteristic of apoptosis (fig. ) in three mammalian cell (vero, hela and cho) and two avian cell lines (pck and cef), but not in hep- cells. the most sensitive line to vt y was vero cells (fig. ) , probably due to receptor speci¢city of the cells, as it is known that variants of vt are more cytotoxic to vero cells than hela cells [ ] ; the receptor for vt and vt is globotriosyl ceramide (gb ) [ ] , whereas the preferred receptor for vt v is globotetraosyl ceramide (gb ) [ ] . it will be interesting to study the receptor for this toxin on the avian cells pck and cef, since vt y is produced by avian e. coli strains. we also observed the presence of multiple vacuoles in the cytoplasm of these cell lines (fig. c) , that simultaneously showed apoptotic features induced by vt y cytotoxin. the microscopic nuclear fragmentation and incorporation of nucleotides shown by the tunel method occurred following vt-treatment of vero cells (fig. ) . by performing the ldh assay we veri¢ed a similar time-response using cd vt y; marked apoptosis e¡ects were also observed when cells were assessed using a toluidine blue assay. based on these data, we suggest that the vt y-induced cell death occurs by the apoptotic pathway after plasma membrane damage. interestingly morphological evidence of apoptosis was observed within min and was maximal within h of toxin administration to vero cells, which is considerably more rapid than previously described for apoptosis induced by vt in epithelial cells [ , ] . ed in pigs is caused by the toxin variant vt v produced by e. coli [ ] . this disease is characterized by anorexia, edema of the eye-lids and neurological abnormalities, such as lack of coordination and paralysis [ ] ; these characteristics, are similar in some respects to those shown by chickens with shs [ ] . here we observed that vt v induced apoptosis in vero cells, but in contrast to vt y the morphological alterations of apoptosis were detected h after vt-treatment (data not shown). from this evidence, a histopathological study of the action of vt y in vivo is necessary to determine whether apoptosis as a vt-mediated mechanism of cell damage is a major feature in the pathogenesis of shs. a simple quantitative procedure using monolayer cultures for cytotoxicity assays (htd/nr- ) a rapid method for the quanti¢cation of microgram quantities of protein utilizing the principle of proteind ye binding e¡ects of escherichia coli shiga-like toxins (verotoxins) in pigs characteristics of the shigalike toxin produced by escherichia coli associated with porcine edema disease verocytotoxin- induces apoptosis in vero cells the association between hemolytic uremic syndrome and infection by verotoxin-producing escherichia coli apoptosis : a basic biological phenomenon with wide-ranging implications in tissue kinetics induction of apoptosis in human renal proximal tubular epithelial cells by escherichia coli verocytotoxin in vitro glycolipid binding of puri-¢ed and recombinant escherichia coli produced verotoxin in vitro puri¢cation and characterization of an escherichia coli shiga-like toxin ii variant. infect. immun escherichia coli strains isolated from pigs with edema disease produce a variant of shiga-like toxin ii signaling and chromatin fragmentation in thymocyte apoptosis experiments in molecular biology evaluation of cytotoxicity in cultured cells by enzyme leakage swollen head syndrome in broiler chicken attempts to reproduce swollen head syndrome in speci¢c pathogen-free chickens by inoculating with escherichia coli and/or turkey rhinotracheits shiga and shiga-like toxins s|ndrome de cabeza hinchada (sh). etiolog|a y pro¢laxis cytotoxin produced by escherichia coli isolated from chickens with swollen head syndrome (shs) caracterizac°a ¬o de uma citotoxina produzida por amostras de escherichia coli de origem aviäria shiga toxin gene in e. coli from swollen head syndrome in chickens. th international symposium and workshop on`shiga toxin (verocytotoxin)-producing escherichia coli infections observations on swollen head syndrome in broiler and broiler breeder chickens comparison of the glycolipid receptor spe-ci¢cities of shiga-like toxin type ii and shiga-like toxin type ii variants toxin-induced cell lysis: protection by -methyladenine and cycloheximide vero cell toxins in escherichia coli and related bacteria: transfer by phage and conjugation and toxic action in laboratory animals, chickens and pigs verotoxins induce apoptosis in human renal tubular epithelium derived cells we thank dr. hernandes f. carvalho for the analysis of cytopathic e¡ects, a. stella degrossoli for technical assistance and paulo yoshio inoki for photographic facilities. we are grateful to fort dodge laboratories, inc. for donating the cef cells. this work was supported by grants from fapesp (fundac°a ¬o de amparo a © pesquisa do estado de sa ¬o paulo). key: cord- -ap ro me authors: oosterhoff, dinja; van de weerd, gerard; van eikenhorst, gerco; de gruijl, tanja d.; van der pol, leo a.; bakker, wilfried a. m. title: hematopoietic cancer cell lines can support replication of sabin poliovirus type date: - - journal: biomed res int doi: . / / sha: doc_id: cord_uid: ap ro me viral vaccines can be produced in adherent or in suspension cells. the objective of this work was to screen human suspension cell lines for the capacity to support viral replication. as the first step, it was investigated whether poliovirus can replicate in such cell lines. sabin poliovirus type was serially passaged on five human cell lines, hl , k , kg , thp- , and u . sabin type was capable of efficiently replicating in three cell lines (k , kg , and u ), yielding high viral titers after replication. expression of cd , the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed cd . furthermore, we showed that passaged virus replicated more efficiently than parental virus in kg cells, yielding higher virus titers in the supernatant early after infection. infection of cell lines at an moi of . resulted in high viral titers in the supernatant at day . infection of k with passaged sabin type in a bioreactor system yielded high viral titers in the supernatant. altogether, these data suggest that k , kg , and u cell lines are useful for propagation of poliovirus. vaccines are pharmacological formulations that incorporate the disease-causing agent or an antigen derived from this agent, which are capable of inducing an immune response once administered to a healthy individual, without causing the disease itself. licensed vaccines can be divided into viral and bacterial vaccines, and viral vaccines can be further classified into four categories: live attenuated viruses, inactivated viruses, subunit vaccines, and virus-like particles. for the production of the first two categories, large amounts of viral particles are needed, and most of these viral vaccines are produced by infecting susceptible cell lines. since there is no standard cell line that can be used for the replication of every virus, a whole panel of different cell lines has been used for vaccine production processes throughout the years. cell lines that have historically often been used for the production of viral vaccines are mrc- and wi- [ , ] . these two cell lines are human diploid cell lines derived from fetuses, and these cells were used for the manufacture of a number of vaccines, for example, hepatitis a, polio, and rubella [ ] [ ] [ ] . diploid cell lines have a finite lifespan and in these cell lines the chromosomes are paired. often these cells retain many characteristics of the cell types from which they originate. the disadvantage of diploid cell lines lies in the fact that the cells can only be cultured for a limited number of passages before the cells die of senescence. in general, diploid cells grow as adherent cells and require serum-containing growth media to grow efficiently. the major benefit of diploid cells is the fact that the cells are nontumorigenic and therefore are considered safe to use for the production of vaccines (reviewed by hayflick et al. [ ] ). given the high demand of vaccines and the restrictions associated with the use of diploid cell lines, in the last decades, continuous cell lines were introduced in vaccine production processes. from a vaccine production point of view, the characteristic of continuous growth is beneficial, since such cells have the potential for an infinite lifespan, and characterized and approved master and working cell banks can be established. a thorough understanding of the cell substrates with respect biomed research international to identity, stability, purity, tumorigenicity, and the presence of adventitious and endogenous agents is, however, essential for the production of quality assured vaccines [ ] . the first continuous cell line approved for the production of vaccines was the vero cell line, originating from african green monkeys and developed at the chiba university in japan. the mechanism of immortalization of vero cells is unknown. it has been described that vero cells at passages to are not tumorigenic in immunocompromised mice [ ] [ ] [ ] and at those passages vero cells are currently used for the manufacturing of viral vaccines. a recent paper, however, concluded that the transition from nontumorigenic to a tumorigenic phenotype of vero cells did not occur until passage [ ] . vero cells have, over the years, proven to be safe, since millions of vaccine doses produced on vero cells have been given to healthy individuals. a major advantage of vero cells is that the cells are sensitive to infection with many different viruses [ ] , meaning that vero cells can be used for the production of a number of different vaccines [ ] [ ] [ ] [ ] . this wide infectivity may be the result of a defective antiviral interferon response of susceptible cells, which was demonstrated in general for cells that are permissive for poliovirus replication [ ] . however, not all viruses are capable of replicating on vero cells and the consensus is that the current repertoire of cell substrates is inadequate for the manufacture of certain types of (new) vaccines. to address this limitation, the vaccines and related biological products advisory committee meeting (vrbpac) recognized in that (human) tumor-derived cell lines could be an important addition to the repertoire of cell substrates for the production of viral vaccines (http://www.fda.gov/downloads/ advisorycommittees/committeesmeetingmaterials/blood-vaccinesandotherbiologics/vaccinesandrelatedbiological-productsadvisorycommittee/ucm .pdf). in some cases, even the only susceptible cell available to propagate specific viruses for which vaccines are needed could be tumor cells. therefore, currently several tumor cells lines are being explored for their capacity to propagate viral vectors, like the madin-darby canine kidney (mdck) cell line [ ] , hela cell line [ , ] , and the per.c cell line of which the latter was immortalized by transfection with adenoviral e proteins [ ] . at the present time though only a limited number of vaccines that were produced in tumorigenic cell lines have entered clinical trials or were registered [ ] [ ] [ ] [ ] [ ] . overall, it can thus be stated that the regulatory opinion on cell lines that are used for the production of viral vaccines has changed radically at the last years with respect to the risks and benefits of immortalized tumorigenic cell lines [ , ] . this is most likely also due to the development of techniques that can detect adventitious viruses or host cell dna in vaccines with a very high sensitivity [ ] . in the future, a potential use of human tumor cell lines for the production of viral vaccines can be foreseen. a characteristic of viruses is that viruses evolve, and due to mutations in their genetic material or recombination with other viruses, outbreaks of dangerous and potential lethal new viruses can occur. in these cases, fast development of vaccines is essential for global health. it would be of great importance if cell lines that are needed for the production of such vaccines could be selected upfront, at the start of a viral outbreak, based on scientific understanding instead of trial and error. in this study, we have made a start with the characterization of human tumor cell lines and their capacity to support viral replication. five human cell lines, capable of growing in suspension and often used in research, but not currently qualified for vaccine production purposes, were selected, hl , k , kg , thp- , and u [ ] [ ] [ ] [ ] [ ] . these cell lines are all cancer cell lines and originally derive from patients with leukemia or lymphoma. all different cell lines originate from blood cells that were abrogated in their development at different promonocytic stages. depending on the mix of cytokines and/or growth factors added to the cells, the cells can differentiate towards several endstages. since the stage of differentiation of a cell can have an effect on susceptibility of the cell for replication of specific viruses, this could be an interesting feature of the selected cell lines [ ] [ ] [ ] . possibly, the cells can become infected as progenitor cells, whereas infection is not possible when the cells are differentiated or vice versa. interestingly, k cells are currently used as a vaccine for patients with lung cancer. irradiated k cells, transfected with the gene encoding gm-csf and cd ligand, were mixed with allogeneic tumor cells and this vaccine was tested in a phase ii trial in lung cancer patients [ ] . the overall aim of this study is to generate data that will facilitate decision making on which substrate may be the best for the production of novel vaccine strains. as a first step for this, we investigated whether poliovirus, as a representative of the picornaviridae family of viruses, can be propagated in the human suspension cell lines. conditions. the human hematopoietic progenitor tumor cell lines hl , u , k , kg , and thp- were cultured in imdm (hl , u , k , and kg ) (invitrogen) or rpmi (thp- ) supplemented with % fetal bovine serum (fbs, paa) and u/ml penicillinstreptomycin (gibco) at ∘ c in a % co humidified atmosphere. the vero cell line was taken along as a positive control cell line and these cells were cultured in virus production serum-free medium (vp-sfm, invitrogen) supplemented with mm glutamine (life technologies) at ∘ c in a % co humidified atmosphere. cho suspension cells, which served as a negative control, were cultured in ex-cell medium (sigma) in shaker flasks (corning) gently rotating at - rpm at ∘ c in a % co humidified atmosphere. to determine whether k cells were capable of growing in bioreactors, cultibags (sartorius stedim) were inoculated with cells at a concentration of . × cells/ml in l of culture medium. cultibag cultures were performed at ∘ c, dissolved oxygen concentration of %, ph . at a constant rocking speed of rpm, and an angle of ∘ . tumor cell lines. to serially passage sabin poliovirus type on the hematopoietic cell lines, × cells were infected biomed research international in t flasks with an moi of . cells and supernatants were harvested if full cytopathic effect (cpe) was observed or after week of culture. after freeze-thawing three times to lyse the cells, samples were centrifuged at rpm to remove cellular debris and half of the supernatant was used to reinfect new hematopoietic cells. this was repeated for times to allow the virus to adapt to the new cell lines in passages. in the last infection round, × cells in a t flask were infected and samples were harvested at day (vero and u ) or day to obtain small viral stocks that were used for subsequent experiments. to determine replication kinetics, the susceptible tumor cell lines were infected with sabin poliovirus type from the parental virus or virus that was passaged for times on the hematopoietic cell lines at moi or moi . , and samples of the supernatant and cellular lysates were harvested at different time points. after harvesting, the supernatant was centrifuged at rpm for minutes to remove cells floating in the medium that did not release their progeny virus yet, and this fraction was added to the cellular fraction. cells still attached to the flask were harvested by scraping the cells from the flask with a cell scraper (becton dickinson) in pbs. after centrifugation at rpm, pbs was removed and the cell pellet was resuspended in ml of pbs. the cellular samples were freeze-thawed three times to release virus from the cells, and after centrifugation at rpm supernatant was transferred to a new tube. the virus titer in all the samples was determined by end-point titration on vero cells. k cells grown in cultibags were infected at an moi of . with passaged sabin poliovirus type when cells reached a concentration of . - . × cells/ml, and samples of the culture medium were taken at days , , , and after infection. samples were filtered ( . m filter) to remove viable cells and the virus titer and d-antigen levels in these samples were determined. to determine the virus titer of sabin poliovirus type that replicated in the different cell lines, the virus titer was determined by end-point titration. in short, adherent vero cells were seeded at a concentration of × cells/ l in -well flat bottom plates in m medium containing % serum. serial -fold dilutions were prepared from cellular lysates or supernatant from hematopoietic cells infected with sabin poliovirus type in serum-containing m medium, and l of these dilutions was added to separate wells. after days, all the wells were scored for the presence or absence of cytopathic effects (cpe), and the virus titer was determined using the reed and muench method, thereby calculating the % cell culture infective dose (ccid )/ml. to calculate the plaque forming unit titer (pfu) from the ccdi value, the ccid was multiplied with . to generate the pfu titer/ml. to be able to compare the number of infectious sabin type polioviruses in the supernatant samples with the cellular lysate samples, the total amount of pfu in the samples was determined by multiplying the pfu/ml titer with the total amount of supernatant or cellular lysate that was harvested. to determine the presence of dantigen in the virus samples, a sandwich elisa was performed as described previously [ ] . briefly, plates were coated with an anti-sabin type caprylated bovine antiserum diluted : in pbs (gibco) overnight at ∘ c. after washing, samples were added for minutes at ∘ c. after washing, a mixture of a sabin type specific mouse monoclonal antibody and an hrp-labeled goat anti-mouse antibody were added for minutes at ∘ c. after four washing steps, the signal reagent highlite was added and the emitted light was detected with a luminometer. to determine the expression of viral receptors on the different cell lines, facs analyses were performed. pe-labeled antibodies directed against human cd (ebioscience), cd (bd pharmingen), and car (millipore) and fitc-labeled anti-human cd (bd pharmingen) were used for flow cytometric analyses, with fitc-or pe-labeled igg antibodies (bd pharmingen) as controls. antibody staining was performed in pbs supplemented with . % bsa and . % sodium-azide for min at ∘ c. after washing of the cells, the stained cells were analyzed on a guava facs (merck millipore) using cell flowjo software. to determine whether hematopoietic cell lines can support replication of sabin poliovirus type , cells were infected with an moi of and cells together with supernatant were harvested at day (for all virus passages in vero cells and for passages - on u cells) or day after infection. after lysis and centrifugation of the cells, half of the supernatant was used for the reinfection of new cells. this procedure was repeated for more times, and virus derived from passage was used for additional experiments. in the samples derived from all passages, the virus titer (ccid /ml) was determined by a virus titration assay. as shown in figure (a) , replication was efficient in the control cell line vero, grown in serum-free medium, yielding high titers of more than × ccid /ml in all passages. in cho cells, sabin poliovirus type could only be detected in the first passage, and this is most likely due to the fact that cells were not washed after primary infection, suggesting that the virus titer is the result of virus that remained present in the culture medium, which was unable to infect the cells. in samples from passages - of sabin poliovirus type added to cho cells, as expected no virus could be detected. a comparable pattern was observed after infection of thp- and hl cells with sabin poliovirus type , suggesting that these two cell lines were refractory for the virus. the k and u cell lines were highly permissive for sabin poliovirus type , resulting in high virus titers in all viral passages. kg cells were also fully permissive for sabin poliovirus type , but the amount of virus obtained after infection of kg cells was slightly lower compared to sabin poliovirus type that replicated in the other permissive cell lines. microscopically it was observed that u cells infected with virus from passages - did not show clear cytopathic effects (cpe) after days of culture, whereas cells infected with virus from passages , , and were harvested after days because full cpe was observed. a photographic overview of cells infected with virus from passage is shown in figure (b) . also, in k cells, clear cpe was visible at day after infection. the amount of virus produced after replication of sabin poliovirus type in k cells did seem to increase after the first passage, suggesting that replication of the passaged virus is more efficient than replication of the parental virus. to be able to compare the d-antigen level per virus, the specific d-antigen level per infectious virus particle was calculated for the fifth viral passage in the permissive cell lines and is shown in figure (c) . for vero cell derived sabin poliovirus type , the d-antigen per infectious virus particle was du/ ccid . in general, it was observed that passaged virus had a d-antigen level per virus that was slightly lower compared to virus replicated in vero cells, varying between . and . du/ ccid depending on the cell line used. hematopoietic cell lines correlated with the capacity to propagate sabin poliovirus type , human cd expression was determined using facs analysis. in figure it can be seen that all hematopoietic cell lines, as well as vero cells, express cd . vero, u , and k had the highest level of expression, whereas the expression level of thp- , kg , and hl cells was lower. also, not all hl cells were positive for cd . since sabin poliovirus type was not capable of replicating in thp and hl cells whereas replication in kg cells was efficient, expression levels of cd thus cannot fully predict the capacity of sabin poliovirus type to replicate in these cell lines. supernatant at day is slightly higher if cells are infected with the passaged virus compared to control virus, although the differences are not significant. it could be that in these cell lines also the replication speed of the passaged virus is slightly enhanced, but differences remain limited because after days the maximal viral titer has been obtained, due to lysis of all the cells. furthermore, it can be concluded from these data that, for all three hematopoietic cell lines tested, the virus titer of the passaged virus at day in the supernatant is comparable to the virus titer obtained days after replication of sabin poliovirus type in vero cells. also, the virus titer in the culture medium remained stable during the days of the experiment. altogether, these data indicate that, with respect to the viral kinetics, all three human hematopoietic cancer cell lines are efficiently producing poliovirus particles. , or k with a low moi. for vaccine production purposes it is important that, after infection of cells with a low moi, a high viral yield in the culture medium can be achieved. to investigate this, cells were infected with an moi of . of passaged sabin poliovirus type , and supernatant and cellular lysates were harvested separately at day and day . the total viral titer in these samples was determined and is shown in figure . in the supernatant of all cell lines tested, at day , a high virus titer, comparable to sabin poliovirus type replicated in vero cells, was observed in the culture medium, indicating that virus replication was efficient during multiple rounds of replication. in k cells, the virus titer was moderately further increased at day after infection, whereas in the other cell lines the virus titer at day in the culture medium slightly decreased compared to the titer at day . interestingly, at both day and day after infection, a high amount of virus was still present in the viable cells. this was also observed in vero cells, and it suggests that not all cells were lysed at day or day by sabin poliovirus type . sabin poliovirus type . to determine whether it is possible to produce sabin poliovirus type at a larger scale, k cells were grown in cultibags. cells were inoculated at a concentration of . × cells/ml in l culture medium. after days, in which the k cells grew to a concentration of . - . × cells/ml, the cells were infected with the passaged sabin poliovirus type at an moi of . . at days , , , and , the cell viability was determined and samples of the culture medium were taken, in which the virus titer and d-antigen concentration were determined. as can be seen in figure (a), the virus replicated efficiently, resulting in titers of × ccid /ml in the culture medium already at day after infection, which remained stable for at least the following days. the d-antigen level at day after infection ( figure (b) ) was somewhat lower than that at later time points. also, a large variation in the d-antigen level between the experiments was observed at day , suggesting that not all viruses expressed d-antigen yet. however, at days , , and after infection, d-antigen levels were high and comparable in all three experiments. hematopoietic tumor cell lines. to investigate whether the human suspension cell lines express multiple viral receptors, surface staining of cd , car, and cd was performed. cd , car, and cd are the receptors for rhinovirus, coxsackie, adenovirus, and hepatitis c virus, respectively. in figure , the percentage of cells expressing the specific receptor is shown. all cell lines, except hl , highly expressed cd and cd . hl cells did express high levels of cd , but cd was only expressed by % of the cells. with respect to car expression, differences between the cell lines were observed. a minority of k cells expressed car, whereas, for the other cell lines tested, - % of the cells did express car. whether the expression of these receptors predicts the capacity of the virus to replicate in these cells remains to be determined. high vaccination rates have helped to prevent many infectious diseases and resulted in less sickness and millions of lives saved (reviewed by rappuoli et al.) [ ] . among the greatest success stories are the eradication of smallpox virus, rinderpest, and the polio eradication program, where poliovirus type was already eradicated in [ ] [ ] [ ] . complete eradication of poliovirus is, however, more difficult than what has been initially anticipated, because of persistence of wild type poliovirus transmission and recurring outbreaks in polio-free countries (reviewed by wassilak et al.) [ ] . the capacity to develop vaccines that induce efficient immune responses in a short period of time, together with a large global immunization rate, is thus essential to prevent viral outbreaks or further spread of viruses. in the last decades, many different cell lines have been used in vaccine production processes, and new cell lines are still being developed. since regulatory views are changing with respect to the risks and benefits of cell lines (reviewed by hess et al.) [ ] , it is important to compare different cell lines for their capacity to propagate specific viruses. with such an approach, using viruses that belong to different viral groups, it might be possible in the future to select producer cell lines based upon scientific understanding instead of trial and error, and timelines needed to produce viral vaccines might be shortened. in this study, we performed a first step for such a comparison, with a focus on human suspension cell lines. these cell lines have an infinite lifespan and this, together with the fact that these cells grow in suspension, could facilitate vaccine production processes. all cell lines described in this study have been used extensively in experimental research. the disadvantage of these cell lines, obviously, is that these cell lines consist of tumor cells that are grown in serum-containing medium and are currently thus not qualified as suitable vaccine substrates. however, with changing regulatory opinions, it is foreseen that continuous cell lines will be accepted for the production of viral vaccines in the (near) future. until then, studies, like this, can be used to gain knowledge about the interaction of cell lines with viruses and/or the development of new producer cell lines that are approved for the production of viral vaccines. because of the experience that our group has with poliovirus production processes [ ] [ ] [ ] , it was decided to perform this study with sabin poliovirus type as the first model virus. first, it was determined whether the five selected cell lines were susceptible for poliovirus infection. k , kg , and u cell lines appeared to be capable of supporting replication of sabin poliovirus type , whereas hl and thp- were not. all five cell lines did express cd , the receptor for poliovirus entry. this means that receptor expression does not predict the capacity of a virus to replicate in a cell. possible explanations for the lack of replication in the cd expressing hl and thp- cells could be that (i) cd expressed on these cell lines is nonfunctional, (ii) the nonsusceptible cell lines lack the expression of a coreceptor on the cellular membrane, or (iii) hl and thp- express an intracellular viral restriction factor. since the tested cell lines have the capacity to differentiate towards several end-stages and it has been shown for other viruses that the differentiation stage of a cell can affect the susceptibility for a virus [ ] [ ] [ ] , it would also be interesting to determine permissiveness of hl and thp- for sabin type after differentiation of the cells towards different end-stages. in the literature it has been described that human blood cell lines were susceptible for infection with poliovirus [ ] , and that study demonstrated that well-differentiated human blood cell lines are more susceptible to cytopathic effects of poliovirus than the less-differentiated blood cell lines. from the selection of cell lines we used in this study, k cells were the least differentiated, but with respect to viral replication we did not observe a difference between k cells and the more differentiated cell lines. another study compared the replication capacity of mahoney poliovirus on k and u cells to hela cells [ ] . compared to hela cells, poliovirus replicated less efficiently in both k and u cells yielding a -fold and a -fold reduced virus output. finally, a study by benton et al. showed differences between k clones in their response to poliovirus infection, because two out of four k cell lines were killed by the poliovirus, whereas in the two other cell lines persistent infections were established [ ] . in our study, we did not observe a persistent infection of k cells, nor that the poliovirus replicated less efficiently in u cells or the less-differentiated k cells. the discrepancies between the observed replication characteristics in these studies with our study could possibly be explained by the fact that in our study the replication characteristics of passaged sabin type were studied, meaning that the virus has had passages to adapt to the new cell line. we did, however, observe already after the first passage of sabin type in k or u cells a high virus titer. this was the amount of virus present in culture medium together with the virus in the cellular lysates, so the majority of the detected virus could have still been present in the cellular fraction, whereas the other studies determined the amount of virus present in the culture medium only. another difference between these studies was that benton et al. and lopez-guerrera et al. used mahoney strain of type poliovirus, and in our study attenuated sabin poliovirus type was used. in two more recent papers, the replication capacity of sabin types , , and or wild type polioviruses was studied on a panel of different suspension cell lines, deriving from humans, avians, or canines. in both studies the virus was not adapted to the new cell lines, but experiments were directly performed with virus produced on vero cells. vlecken et al. compared a number of adherent and suspension cell lines for the capacity to replicate sabin poliovirus types , , and . of the cell lines tested (bhk- , cho-k , cap, smdck, and age .cr.hs), only the cap cell line was capable of propagating sabin type polioviruses, whereas all the other suspension cell lines were not capable of supporting viral replication [ ] . in the second study, published by sanders et al., the capacity of per.c to support replication of brunenders, mef- , and saukett poliovirus was determined in comparison to vero cells [ ] . per.c is an immortalized human cell line capable of supporting the replication of a number of viruses, like influenza virus and west nile virus [ , ] . the poliovirus was capable of replicating efficiently in the per.c cell line, resulting in a high virus yield, which can be attributed to the fact that per.c cells can be cultured under optimized conditions to very high cell densities. altogether, these two studies suggest that, for efficient replication of poliovirus, human or primate cells are needed, since, also in the adherent cell lines tested by vlecken et al., viral replication was not observed in cell lines with another origin [ ] . this observation underscores the need of a wider panel of human/primate cell lines for the production of viral vaccines for human diseases. in our study, we have identified three additional cell lines that are susceptible for infection and replication of poliovirus that can also be grown at a larger scale in a disposable bioreactor system (only tested for k cells). it is important to realize that, by passaging a virus on a new cell line, the virus can adapt to this new cell line to optimize its replication cycle. this can also be accompanied by changes in antigenicity or virulence of the adapted virus. a new combination of cell line and virus should thus always be analyzed thoroughly, before the new cell line can be used for the production of viral vaccines. in this study, we did observe a difference in replication speed between original virus and virus passaged on kg cells, but sequencing is needed to determine whether the virus truly adapted to the cell line. since we already observed that also other sabin poliovirus types are capable of replicating in the permissive three cell lines (oosterhoff et al., unpublished data) and that all cell lines express other viral receptors, needed for efficient infection with rhinovirus, coxsackie, adenovirus, and hepatitis c virus, it would thus be interesting to determine the capacity of these viruses to replicate in these cell lines. ultimately both the panel of (human) cell lines and viruses need to be expanded, in order to be able to predict upfront the suitability of cell lines for the production of specific viral vaccines, based on scientific understanding, thereby reducing timelines and costs in vaccine production processes. in conclusion, these data demonstrate that k , kg , and u cell lines are efficient in supporting the replication of sabin poliovirus type . all five human hematopoietic cell lines expressed cd , which thus did not explain susceptibility to viral replication, although we did not study the functionality of cd . furthermore, we have shown that in kg cells the passaged sabin poliovirus type replicated more rapidly than the control virus. infection of k , kg , and u at an moi of . resulted in a high viral titer in the culture medium after days. also, k cells grown and infected with adapted sabin poliovirus type in a disposable bioreactor system yielded high viral titers in the culture medium. altogether, it is concluded that k , kg , and u cell lines can be used for the propagation of poliovirus and in follow-up studies the capacity of other (entero)viruses to replicate in these cell lines will be determined. characteristics of a human diploid cell designated mrc- preparation of poliovirus vaccines in a 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infection of established human blood cell lines: relationship between the differentiation stage and susceptibility or cell killing restriction of poliovirus rna translation in a human monocytic cell line k cell strains differ in their response to poliovirus infection comparison of initial feasibility of host cell lines for viral vaccine production per.c cells as a serum-free suspension cell platform for the production of high titer poliovirus: a potential low cost of goods option for world supply of inactivated poliovirus vaccine the human cell line per.c provides a new manufacturing system for the production of influenza vaccines safety and efficacy in geese of a per.c -based inactivated west nile virus vaccine the authors declare that there is no conflict of interests regarding the publication of this paper. key: cord- -jq xumrh authors: postnikova, elena; cong, yu; dewald, lisa evans; dyall, julie; yu, shuiqing; hart, brit j.; zhou, huanying; gross, robin; logue, james; cai, yingyun; deiuliis, nicole; michelotti, julia; honko, anna n.; bennett, richard s.; holbrook, michael r.; olinger, gene g.; hensley, lisa e.; jahrling, peter b. title: testing therapeutics in cell-based assays: factors that influence the apparent potency of drugs date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: jq xumrh identifying effective antivirals for treating ebola virus disease (evd) and minimizing transmission of such disease is critical. a variety of cell-based assays have been developed for evaluating compounds for activity against ebola virus. however, very few reports discuss the variable assay conditions that can affect the results obtained from these drug screens. here, we describe variable conditions tested during the development of our cell-based drug screen assays designed to identify compounds with anti-ebola virus activity using established cell lines and human primary cells. the effect of multiple assay readouts and variable assay conditions, including virus input, time of infection, and the cell passage number, were compared, and the impact on the effective concentration for % and/ or % inhibition (ec( ), ec( )) was evaluated using the fda-approved compound, toremifene citrate. in these studies, we show that altering cell-based assay conditions can have an impact on apparent drug potency as measured by the ec( ). these results further support the importance of developing standard operating procedures for generating reliable and reproducible in vitro data sets for potential antivirals. ebola virus (ebov) infection in humans and nonhuman primates is often associated with high morbidity and mortality rates, as well as severe hemorrhagic fever [ ] [ ] [ ] [ ] . ebov is a biosafety level- pathogen transmitted by contact with bodily fluids, fomites, or droplets from plos infected patients. ebov is considered a significant threat to public health and global security due to its potential to be used as a bioweapon [ ] [ ] [ ] [ ] . currently, no fda-approved vaccine or therapeutic agents are available, and supportive care remains the standard for ebola virus disease (evd) treatment. therefore, accelerated efforts in the development of therapeutics is a key objective in the ebov research community, especially since the - evd epidemic in western africa. drug discovery and development requires considerable time and resources to identify an effective drug that will progress to clinical trials [ , ] . as a result, research investigating the repurposing of drugs for additional indications have become increasingly more prevalent to accelerate the identification of therapeutic drugs for evd. the off-label use of fda-approved drugs is particularly advantageous as safety concerns and ethical problems have already been addressed [ ] [ ] [ ] [ ] . to effectively identify potential compounds of interest from large libraries of chemical compounds, share more reliable and reproducible data between laboratories, and provide data to the international community, appropriate methods or models need to be established. furthermore, these models should be evaluated to determine how predictive they are for identifying compounds most likely to be efficacious in humans. for evd, indications of efficacy could include successful treatment and survival of patients, alleviation of disease severity, or mitigation of clinical symptoms associated with ebov infection. a variety of methods are available to measure antiviral activity in vitro. however, the development of a screening assay to detect compounds with anti-ebov activity was previously limited due to the difficulty of developing a suitable high-throughput screening system for a biosafety level- viral pathogen. classical methods evaluate drug efficacy include the reduction of virus yield [ ] or a decrease in viral rna transcription as determined by real-time polymerase chain reaction (pcr) [ ] [ ] [ ] . recent therapeutic screening methods have transitioned from the classical methods of measuring viral inhibition to assays with the ability to be automated, resulting in higher-throughput. assay chemistries have been developed to enable the homogeneous measurement of a variety of different endpoints such as cytopathic effect, viral protein or reporter gene expression, which can serve as markers of viral replication [ ] . the growing interest in identifying drugs with activity against ebov has resulted in a variety of assays and readouts for activity as well as cytotoxicity. the use of cell-based assays for highthroughput screening of compound libraries has increased steadily over recent years [ ] [ ] [ ] [ ] . as cell-based assays monitor specific viral proteins and provide the means to screen for potent viral inhibitors intracellularly, these assays identify drug candidates with desired pharmacological properties in the primary drug-discovery pipeline. in this study, the susceptibility to ebov using both immortalized cell lines and primary monocyte-derived macrophages (mdms) was investigated under a variety of conditions such as different multiplicities of infection (mois), times of exposure, and the cell passage numbers. a cell-based assay with ebov vp -specific antibody was used to detect infected cells. fluorescence or chemiluminescence readout was used for determining signal-to-noise (s/n) ratio, and a high-content imaging system was applied to determine the percentage of ebov-positive cells. toremifene citrate, which the world health organization considered evaluating in a clinical trial for treatment of evd, was chosen as a positive control to measure ebov inhibition under each condition [ ] [ ] [ ] [ ] . the conditions under which drugs are tested can influence their apparent potency. while testing drugs for the world health organization (who) community, we received many requests to repeat experiments under specific conditions to confirm activity identified by another laboratory. the resulting data sets indicated just how variable the ec value can be under varying assay conditions. the data presented here provides insight on how different assay parameters can impact the in vitro efficacy of potential anti-ebov antivirals using toremifene citrate as a model compound. vero e (african green monkey kidney; atcc ) cells were obtained from the american type culture collection (manassas, va). vero c (e ) cells (african green monkey kidney, working cell bank nr- ) were obtained through bei resources (national institute of allergy and infectious diseases [niaid] , national institutes of health [nih], manassas, va). huh cells (human hepatocellular carcinoma) were obtained from dr. hideki ebihara (niaid, rocky mountain laboratories, hamilton, mt). all cell lines were maintained at the integrated research facility (irf) following cell source instructions. a primary vero e and huh cells culture were grown to % confluency in a t- (fisher scientific) or triple layer tissue culture flask (nunc) containing dulbecco's modification of eagle medium (dmem) (gibco) supplemented with % heat-inactivated fetal bovine serum (fbs) (sigma). cells were dispersed by trypsin (gibco) treatment and then reseeded into secondary cultures. the process of removing cells from the primary culture, diluting, and then transferring them to secondary cultures constitutes a passage. both cell lines were provided at passages - , at which point a new culture was introduced and the previous passage series was ended. additionally, cell cultures were required to be a least % viable in order to achieve acceptance criteria and to be plated for use in a screening assay. the generation of mdms has been described in previous studies [ , ] . briefly, pbmcs were isolated from human whole blood by density-gradient centrifugation over histopaque ( . g/ml, sigma-aldrich, st. louis. mo). monocytes were purified using human cd -specific microbeads (miltenyi biotec, san diego, ca, - - ) following manufacturer's instructions. cd + monocytes were differentiated into mdms by culturing for - days with recombinant human macrophage colony-stimulating factor (bio-techne, minneapolis, mn, -mc- ) and conditioned medium from kpb-m cells (kind gift from dr. atsunobu hiraoka, scgf research laboratory, kyoto, jp). media were replaced every - days during the incubation for a total of - days. the cells were harvested and plated on desired -well plates day prior to the drug screen assay. the differentiated mdms were characterized by flow cytometry before assay initiation. toremifene citrate (oral solution) tested in this study was purchased from sigma-aldrich (cas - - ; t - mg). the makona isolate of ebov (h. sapiens-tc/gin/ /wpg-c ) (ebov/mak, genbank accession no. kp ), a kind gift from dr. gary p. kobinger (public health agency of canada, winnipeg, ca), was used in these studies. to generate virus stocks, ebov/mak was inoculated at an moi of . in vero c cells (bei resources, manassas, va, catalog nr- ). when the cytopathic effect was visible at day - after infection ( - % of monolayer showing cpe), cell culture supernatants were harvested and clarified by centrifugation. the ebov/mak titer was determined by plaque assay in vero e cells. virus titers were measured using -fold serial dilutions of culture supernatant in triplicate infections of vero e cell monolayers in -well plates. after incubation at ˚c for h (plates were rocked every minutes), ml of medium containing x mem (gibco), . % avicel (fmc biopolymer), and x antibiotic-antimycotic (gibco) were added to each well ( ml/well). after days post-incubation, virus plaques were stained with . % crystal violet (ricca chemical) in % neutral buffered formalin (thermo scientific) and infectivity titers were measured in plaque forming units per ml (pfu/ml). all procedures using live ebov were performed under biosafety level- (bsl- ) conditions. vero e and huh cells were seeded overnight at to × cells per well and mdm cells were plated at × cells per well in μl of dulbecco's modified eagles's medium with % fetal bovine serum in black opaque (thermo fisher scientific, waltham, ma, corning , - - ) or clear bottom -well greiner microplates (greiner bio-one, monroe, nc, ). ebov/mak isolate was diluted in culture media to the specified mois (the titers used to determine moi hereby were generated on vero e cells) in -well plates. the cells were then infected by transferring μl of ebov/mak isolate from the virus dilution plates to cell plates using the -well liquidator (rainin instrument, oakland, ca). the cells were incubated at ˚c and % co for the indicated periods of time. the plates were fixed by adding μl of % neutral-buffered formalin (final concentration %) at , , or h postinoculation (hpi). after fixing for h, the plates were transferred to a bsl- lab for antibody staining as described previously [ ] . briefly, ebov was detected by exposure of the infected, fixed, and permeabilized cells to a monoclonal mouse antibody specific to the ebov vp matrix protein (b-md -bd -ae , made by us army medical research institute of infectious diseases, frederick md under centers for disease control and prevention contract) [ ] , followed by staining with an alexafluor goat anti-mouse igg (heavy + light chains) antibody (life technologies, carlsbad, ca) at ˚c for h. fluorescence was quantified using a tecan plate reader (infinite m , tecan us, morrisville, nc) or an high-content imaging (hci) system (operetta, perkinelmer, waltham, ma). hci images were collected at x magnification using to fields of view in each well to quantify the percent of ebov-positive cells. the viability of the cell layer was monitored by staining cell nuclei with the hoechst dye (molecular probes) at ˚c for h. columbus . . software (perkinelmer) was used to analyze the hci data. the chemiluminescent enzyme-linked immunosorbent assay (celia) was performed by detecting ebov with the anti-ebov vp antibody followed by staining with the horseradish peroxidase (hrp)-conjugated goat anti-mouse secondary antibody (seracare, milford, ma, cat. # - ). chemiluminescence was quantified using pico chemiluminescent substrate (thermo fisher scientific inc., rockford, il) and a plate reader (infinite m tecan). vero e , huh and mdm cells were seeded as described above in μl media overnight at ˚c with % co . compounds in dimethyl sulfoxide were prediluted to reduce dimethyl sulfoxide concentration to . % or lower. compounds were prediluted in dilution blocks before performing a final : dilution by transferring μl of each compound to cell plates containing μl of cell culture media. this dilution achieved a desired compound concentration in μl of cell culture media. the process of performing the drug screen assay is shown in fig . three plates were set up per experiment, two plates (clear bottom -well greiner microplates) for detecting inhibition of ebov, and one mock plate (black opaque plate) for determining drug cytotoxicity. after h of predilution and transport to the bsl laboratory, μl of virus (or mock control) at the desired moi was added to cells. at , or hpi, assay plates were fixed at final concentration of % nbf for h before transferring to a bsl- lab for staining. infected cells were detected as described above. to further confirm the accuracy of assays with high background, chemiluminescence assay was performed afterwards. cytotoxicity in mock infected cell plates was measured or h after treatment with compounds using the celltiter glo luminescent cell viability assay kit according to the manufacturer's instructions (promega, madison, wi). luminescence was read on the infinite m tecan plate reader (fig ) . non-linear regression analysis and curve fitting parameter were performed to calculate ec s, ec s and % cytotoxic concentration (cc s) (graphpad software, la jolla ca) [ ] using dose-response curves for the compounds (toremifene citrate). error bars of dose-response curves represent the standard deviation of three replicates. equations for the ratio of s/n, percentage of ebov-positive infected cell, and z' factor were defined previously [ , ] , and ec s were used as parameters for assay validation. the quality control of cell-based assay is at specific assay endpoints, cells are fixed and transferred to the bsl- . immunostaining was performed with a ebov-specific antibody against vp and a fluorescent or chemiluminescent secondary antibody using a plate washer/dispenser. fluorescence is quantified on a plate reader. the hci system (operetta) is used to detect ebov-positive cells and count cells with a nuclei stain (hoechst ). in parallel, cytotoxicity assays (celltiter glo) with mock infected cells are performed at bsl- . luminescence is read on the infinite m tecan plate reader. data are analyzed using graphpad prism and/or columbus software (operetta). https://doi.org/ . /journal.pone. .g factors that influence ebola antiviral activities in cell-based assays plos one | https://doi.org/ . /journal.pone. march , represented by z' factor which is defined as equation z' = [ -(( Ã sd pos )+( Ã sd neg ))/(imean pos -imean neg )]. in our assay, the z' criteria is as follows: z' = . - . corresponds to an excellentassay; z' = - . corresponds to a suboptimal assay; z' < corresponds to a unsuccessful assay [ ] . the susceptibility to ebov infection was evaluated in multiple cell types in cell-based assays measuring anti-ebov activity. vero e , huh and mdm cells were infected with ebov/ mak isolate at different mois, and the assay was terminated after , , or hpi. cells were stained with a fluorescent antibody, and hoechst dye was used to visualize the cell nuclei. infectivity was measured using the high-content imaging (hci) system as percentage of vp positive cells. the growth of ebov/mak isolate in three cell types was compared over time. in vero e cells, ebov spread slightly slower, and the number of positive cells were overall lower compared to huh cells (fig a- d ). mdms were the most susceptible to ebov among the cell types. at hpi, mdms already exhibited a typical dose response relative to virus input, while only minimal ebov replication was observed in vero e and huh cells at hpi at all mois tested. virus spread effectively at hpi with higher ( to . ) moi of vero e , huh cells and most of mois in mdms (fig a- f ). the infection in mdms was saturated at hpi at almost all mois (fig e and f ). the nuclear stain for all cell types showed that the cell layer deteriorated with increased virus inoculum and duration of infection as was clearly evident at the hpi time point and at higher virus input (mois of . ) (fig b, d , f and g). the cell layer frequently became fragile, and at later time points, the cell layer would lift off the well surface possibly because of increased exposure to virus, higher moi, or manipulation of plates during the fixing/staining procedure (fig g) . the hci system proved to be invaluable for determining optimal assay conditions (end point and virus input) and in identifying potential issues such as cell layer integrity. different virus input and endpoints were assessed to identify an acceptable range for testing drugs in vero e and huh cells (tables and ). the same experimental plates from fig a- d were used for this analysis. the fluorescence s/n ratio was calculated using a tecan plate reader, and the percentage of ebov-positive cells was determined using hci. for reproducible data, we determined that the percentage of ebov-positive cells should be kept within a range of to % and the s/n ratio at to . at lower s/n ratios (< ) or lower percentage of infected cells (< %) distinguishing activity of a drug from non-activity becomes increasingly difficult. on the other hand, at higher s/n ratios (> ), when cell infection rate trends towards - % and cells overgrow at later time points, the risk of a disrupted cell layer is greater. cell layer disruption increases the variability of the assay. therefore, the optimization of assay conditions (i.e., duration of infection, virus input) was carefully calibrated resulting in a compromise between signal strength and cell viability. based on the data obtained from fig and tables and , two optimized conditions for the fluorescence assays were selected. an moi of with hpi as endpoint and an moi of . with an endpoint of hpi produced a robust signal for all cell types, vero e , huh and mdms (fig a- f , tables and ). vero e cells were infected with ebov at around . %, and mdms and huh cells usually had a stronger signal with around % or higher of ebov-positive cells (fig c- f ). the two conditions had a similar signal or ebov infection during the assay development phase, vero e cells appeared less reliable in producing a consistent viral spread, and this lack of reliability appeared to be due to cell culture passage history. to address this matter, vero e and huh cells were analyzed at different cell passage numbers by determining percentage of ebov-positive cells (fig a and b ). both cell types were infected with a moi of for h. the viral spread of huh cells were consistent over cell culture passages - , indicating that the passage number does not impact ebov spread c the percentage of ebov-positive cells was determined with a vp /alexa- antibody stain to detect ebov and a nuclear stain to quantify the number of cells in a high-content imaging system. d s/n is the ratio of the fluorescent signal (mean from infected wells) to the noise (mean from mock-infected wells) quantified using a plate reader. abbreviations: ebov + , ebola virus-positive; hpi, hours post-inoculation; moi, multiplicity of infection; s/n, signal-to-noise ratio. in huh cells in the range tested. in contrast, ebov spread on vero e cells varied considerably between cell passages - with peak infection (replication efficacy) at cell culture passages - ( fig a) and equally lower for early or late passages. significant differences were observed at passages , , , , and when compared to the passage or using an ordinary one-way anova following dunnett's multiple comparison in graphpad prism . . occasionally, exceptions to this trend were observed (fig ) . the infectivity of ebov in vero e and huh cells were compared at an early (p ) and a late passage number (p ) at a range of mois ( . - . ) for h. in this case, vero e cells tested at the later passage showed a lower infection rate (< %) as expected, while the early passage demonstrated an unusually high infection rate (up to %). infectivity in huh cells remained consistent regardless of passage number, vero e cells were inconsistent overall despite the trend originally observed. both the virus input and duration of the experiment can have an impact on drug activity. to address this, we evaluated the in vitro efficacy of toremifene citrate, an fda-approved drug with proven anti-ebov activity [ ] , under different parameters using fluorescence as the read out. huh cells were infected with a constant moi of and treated with toremifene citrate for , , or h (fig a) . later time points resulted in a decrease in activity with the ec at hpi . -fold higher than at hpi. when the time point remained constant at hpi the activity of toremifene citrate increased as the virus input decreased (mois of . , . , , or ) (fig b) . the ec at a moi of . was . -fold lower than at a moi of . in addition to time and virus input, the cell type used in the assay can result in differences in the calculated ec . anti-ebov activity of toremifene citrate was measured using variable assay endpoints ( , and hpi), mois ( . , . , . , and ), and cell lines (vero e and huh cells, fig ) . ec values for toremifene citrate increased with exposure time and virus input in both cell types. overall, activity in vero e cells was higher with maximum activity (ec = . μm) at hpi and an moi of . . in huh cells, maximum activity (ec = . μm) was detected at hpi and an moi of . . ec values for toremifene citrate increased with exposure time and virus input in both cell types indicating that more drug is required to produce the same anti-ebov effect. factors that influence ebola antiviral activities in cell-based assays to increase sensitivity of the drug screen assays, a chemiluminescent enzyme-linked immunosorbent assay (celia) was developed for evaluating compounds for anti-ebov activity. the celia combines the advantage of specificity of an immunoassay with the high sensitivity of a chemiluminescent enzyme detection assay and is a simple and low cost screening assay [ , ] . the celia was compared to the fluorescent assay (detected by regular plate reader or the hci system) by testing the efficacy of toremifene citrate against ebov infection in huh cells with a range of mois ( . , . , , and ) at three different time points ( , and hpi) . assay parameters, signal-to-noise (s/n), and z' factor, were compared between the assays (table ) . at the earliest time point ( hpi), the celia had higher sensitivity than the fluorescent detection assay as the z' were in the acceptable range (> . ) even at the lowest moi of . . in contrast, the fluorescent assay required higher mois for reliable data sets (z'> . ). at the hpi time point, both detection methods generated high quality data sets with the celia providing improved z' factors (> . ). at hpi, the celia and the fluorescent assay also produced excellent data using hci for detection, while the fluorescent data detection by regular plate reader showed higher variability, maybe due to cytopathic effects. the ec s and ec s factors that influence ebola antiviral activities in cell-based assays were determined on those data sets with acceptable z' (> . ) and overall there was good correlation between the different detection methods (table ). all detection assays demonstrated that higher moi and longer times of exposure led to an increase of the ec values for toremifene citrate. in contrast, the ec values showed considerably lower fluctuations under the different conditions tested. the established celia was used to compare anti-ebov activity of toremifene citrate in different cell types, vero e , huh and mdms using an moi of . and a time point of h (fig ) . huh cells and mdms had s/n ratios in the range of s with z' factors generally above . (fig a) . the s/n for vero e cells ranged from > to over , leading to more variability as reflected in the wider range of z' factors ( . - . ) (fig a) . the activity (ec ) of toremifene was compared, and considerable differences were detected for the cell types ( fig b) . the efficacy of toremifene citrate was -fold higher in huh cells than in mdms, and -fold higher than in vero e cells (fig b) . in summary, the chemiluminescent drug screen assay for evaluating anti-ebov activity of compounds is a very robust, reliable and reproducible method. the data presented in this study exemplify the importance of having guidelines for testing drugs in vitro for antiviral activity and generating reproducible data sets that can be shared and confirmed by outside laboratories. several parameters should be considered when testing factors that influence ebola antiviral activities in cell-based assays drugs in vitro for antiviral efficacy. cell type, assay endpoint, and the virus input are among the most important factors [ ] . the vero e cell line, derived by immortalization of african green monkey kidney cells, is the most commonly used cell line for testing antivirals against filoviruses. ebov propagates very well in this cell line, and many laboratories, including ours, generate their virus stocks and determine virus titers in vero e cells. however, the argument to use a cell line with more relevance to human disease has come up repeatedly. hence, the human liver cancer cell line, huh and human mdms were chosen for these studies. macrophages are relevant to human disease, and they are considered to play an important role in virus dissemination in pathogenesis of evd [ ] . therefore, a drug that is highly effective at inhibiting ebov infection in mdms may better translate to in vivo potency than vero e cells. the susceptibility of three cell types was compared over a range of virus input and over time. all cell types were permissive to ebov infection, but to different degrees. mdms demonstrated optimal replication starting as early as hpi at an of moi . . in contrast, ebov grew slower in vero e and huh cells. the optimal conditions for evaluation of drug efficacy using the fluorescent assay were at an moi of . for the h time point or at an moi of for the h time point for all cell types to ensure a strong virus signal and avoid destruction of cell layer (fig g) . however, an moi of is not suitable for detecting inhibitors of later steps in the virus life-cycle. the ability to detect inhibitors of virus assembly or egress will be reduced with increasing moi, as higher proportions of the cells will be infected even in the factors that influence ebola antiviral activities in cell-based assays though vero e cells are one of the most broadly used cell lines for testing compounds in in vitro assays, our data show that the performance of these cells is not ideal especially when evaluating drugs for anti-ebov activity. although it is never a good idea to passage cells for too long [ , ] , we found that at lower or higher passages the vero e cells can show a decreased percentage of ebov-positive cells. huh cells showed more efficient and more reliable virus replication irrespective of passage number. immortalized cancer cell lines are in general an easy choice for in vitro drug testing because they are easy to handle, propagate, expand, and plate on a week-by-week basis with relative consistency. in contrast, primary cell types such as human primary macrophages do not proliferate in cell culture and are ideally generated fresh from human blood upon use. for large scale testing, procuring the blood volumes needed for generating mdms from a cumbersome isolation technique could be a challenge. mdms demonstrated very efficient spread of virus through cell culture over time. donor-todonor variability precludes consistency, and in general, macrophages from different donors are not pooled. despite of the constraints, testing a selection of drugs that are closer to human clinical studies in human macrophages makes sense to get a better idea on efficacy in a more relevant target cell. fluorescence was detected by two different read outs and each technique has its advantages. the plate reader will measure the average fluorescence across the whole well. readings are quick and conducive to handling large numbers of plates during screening. however, the quality of the cell layer cannot be monitored. hci provides images of or more fields of each well, and a nuclear stain can be added in parallel to indicate the viability of the cell layer. in the assay development phase, this tool is especially useful in identifying conditions that will ensure a healthy cell layer or identify issues such as plate corner or edge effects for assay quality control. one drawback is that not the whole well is imaged, but only a certain number of fields inside the well. it is important to pick the fields wisely to avoid bias or skewing of data. scanning at least fields per well is recommended for statistical purposes, but that will increase reading time per plate. the time to read and analyze the hci data can take considerably longer than the data acquired with the regular plate reader. hci measures different parameters such as percentage of ebov-positive cells and mean signal intensity per cell. both the regular plate reader and hci have unique applications in a drug testing program. while a plate reader is good for a quick readout of large number of plates, the hci system is used to cross check on quality of cells and to clarify issues of noise and signal variability. in addition to the fluorescent assay, we also implemented a celia using an hrp-labeled antibody, which amplified the signal and increased the sensitivity of virus detection. as expected, the celia showed an improvement in the quality of data sets compared to the fluorescent assay. s/n ratio and z' factor were in an acceptable range as early as hpi with the lowest virus input detected at an moi of . . an ec could also be determined at this time point. at or hpi, the two fluorescent read outs were similar to the chemiluminescent read out. toremifene citrate is a selective estrogen receptor modulator reported to be active in vivo against ebov in mice [ , ] . this is an fda-approved drug for treating breast cancer [ ] . mechanism of action studies showed that toremifene citrate affects ebov virus entry at the stage of virus-fusion with the endosomal membrane [ ] . toremifene citrate was chosen by the who as a potential candidate for clinical evaluation in evd patients. we evaluated the performance of toremifene citrate on anti-ebov activity using different conditions. a trend towards higher ec s with increasing input virus and longer assay time was observed. the data indicate that if the amount of virus present in cells or tissues is high enough, the drug will be less active. also with longer time for the virus to replicate, drugs may be unable to stem high viral replication. in contrast, the ec values were less affected by high moi or longer time points, which may, therefore, be a more reliable parameter for comparing data sets between laboratories. serum in the media and pretreatment of cells with the compound can also have a considerable effect on antiviral activity. toremifene was compared directly to brincidofovir under a panel of different conditions [ ] showing that activity of brincidofovir was dependent on media conditions with low serum (< %) and h pretreatment before adding virus. in contrast, the serum concentration had no effect on the activity of toremifene citrate, and pretreating cells for h (instead of h) decreased the activity of this drug. many reports on the repurposing of fda-approved drugs discuss and compare a drug's in vitro ec (determined in cell cultures) with its maximum concentration in human plasma (determined in clinical trials) when evaluating the potential of the drug to have in vivo activity. however, our data show that the ec of a drug can range over more than a log based on testing conditions (e.g, moi, time of endpoint, serum). johanson et. al. [ ] reported ec s of . and . μm for toremifene with two different ebov strains ebov/kik and ebov/may, respectively, at hpi with . moi by celia using the c antibody. these data are comparable to the ec of . ± . μm for ebov/mak determined in our assay under similar conditions (table ; moi . / hpi). understanding how assays are performed and the potential variables is important, and caution should be used when using in vitro data for making decisions to advance a drug to in vivo studies. addition, we acknowledge laura bollinger and jiro wada at the irf for technical writing services and figure preparation, respectively, for this manuscript. multiple ebola virus transmission events and rapid decline of central african wildlife ebola virus: from discovery to vaccine exotic emerging viral diseases: progress and challenges phase clinical trial of apical membrane antigen : an asexual blood-stage vaccine for plasmodium falciparum malaria the soviet union's anti-agricultural biological weapons 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toxin, and plating density in cell-based cytotoxicity assays cytotoxicity testing: measuring viable cells, dead cells, and detecting mechanism of cell death inhibition of ebola virus entry by a c-peptide targeted to endosomes the lipid moiety of brincidofovir is required for in vitro antiviral activity against ebola virus we thank irf cell culture staff in preparing the cells used in this study. we thank dr. atsunobu hiraoka (scgf research laboratory, kyoto, jp) for the kind gift of conditioned medium from kpb-m cells. we thank dr. gary kobinger (public health agency of canada, winnipeg, ca) for the makona isolate of ebola virus (genbank accession no. kp ). we thank dr. hideki ebihara (niaid, rocky mountain laboratories, hamilton, mt) for huh (human hepatocellular carcinoma) cells. we thank dr. sheli radoshitzky (usamriid, frederick md) for her gift of vp ebov vp matrix protein (b-md -bd -ae . in key: cord- -gqbtkf x authors: lee, sunhee; kim, youngnam; lee, changhee title: isolation and characterization of a korean porcine epidemic diarrhea virus strain knu- date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: gqbtkf x severe outbreaks of porcine epidemic diarrhea virus (pedv) have re-emerged in korea and rapidly swept across the country, causing tremendous economic losses to producers and customers. despite the availability of pedv vaccines in the domestic market, the disease continues to plague the korean pork industry, raising issues regarding their protective efficacy and new vaccine development. therefore, pedv isolation in cell culture is urgently needed to develop efficacious vaccines and diagnostic assays and to conduct further studies on the virus biology. in the present study, one korean pedv strain, kor/knu- / , was successfully isolated and serially propagated in vero cells for over passages. the in vitro and in vivo characteristics of the korean pedv isolate were investigated. virus production in cell culture was confirmed by cytopathology, immunofluorescence, and real-time rt-pcr. the infectious virus titers of the viruses during the first passages ranged from ( . ) to ( . ) tcid( ) per ml. the inactivated knu- virus was found to mediate potent neutralizing antibody responses in immunized guinea pigs. animal studies showed that knu- virus causes severe diarrhea and vomiting, fecal shedding, and acute atrophic enteritis, indicating that strain knu- is highly enteropathogenic in the natural host. in addition, the entire genomes or complete s genes of knu- viruses at selected cell culture passages were sequenced to assess the genetic stability and relatedness. our genomic analyses indicated that the korean isolate knu- is genetically stable during the first passages in cell culture and is grouped within subgroup g b together with the recent re-emergent korean strains. porcine epidemic diarrhea (ped) is a devastating disease in pigs that is characterized by acute enteritis and lethal watery diarrhea followed by dehydration with high mortality in suckling piglets (debouck and pensaert, ; saif et al., ; pijpers et al., ) . the disease was initially recognized in england in and has then spread to swine-producing european countries (oldham, ; pensaert et al., ) . since the s, ped has become rare in europe and is more often associated with post-weaning diarrhea in adult pigs (saif et al., ) . ped was first reported in asia in and has since had a great economic impact on the asian pork industry (chen et al., ; kweon et al., ; li et al., ; puranaveja et al., ; takahashi et al., ) . in may , ped outbreaks suddenly appeared in the united states and have rapidly spread nationwide as well as to canada and mexico, causing high mortality in newborn piglets and significant financial concerns (mole, ; stevenson et al., vlasova et al., . the etiological agent of ped, ped virus (pedv), was identified as a coronavirus in , which is a member of the genus alphacoronavirus within the family coronaviridae of the order nidovirales (lai et al., ; pensaert and de bouck, ; saif et al., ) . pedv is a large, enveloped virus possessing a single-stranded positivesense rna genome of approximately kb with a cap and a polyadenylated tail (pensaert and de bouck, ; saif et al., ) . the spike (s) protein of pedv is the major envelope glycoprotein of the virion and plays pivotal roles in interacting with the cellular receptor for virus entry and mediating neutralizing antibodies in the natural host (jackwood et al., ; lai et al., ; lee et al., ) . therefore, the pedv s glycoprotein is known to be an appropriate viral gene for determining the genetic relatedness among pedv isolates and for developing diagnostic assays and effective vaccines (chen et al., ; gerber et al., ; lee et al., ; lee and lee, ; oh et al., ) . the first ped epizootic in korea was confirmed in (kweon et al., ) . however, a retrospective study revealed that pedv already existed as early as (park and lee, ) . since the emergence, ped outbreaks occurred every year, resulting in substantial economic losses to the korean swine industry until early . after severe outbreaks of foot-and-mouth disease (fmd) during to , however, the prevalence of pedv infections was occasional with only sporadic outbreaks in korea. this epidemic situation probably resulted from the mass culling of more than one-third of the entire domestic pig population in korea during the - fmd outbreaks. however, starting in november , severe ped epidemics re-emerged in korea and swept more than % of pig farms lee et al., a,b) . although both modified live and killed vaccines against ped are commercially available in korea, continuous ped epidemics indicate a low effectiveness of the domestic vaccines. this result appears to be due to genetic and antigenic differences between s proteins of vaccine and field strains (lee et al., ; oh et al., ; lee and lee, ) . thus, the lack of effective vaccines enhances the need for the development of next-generation vaccines to control ped. pedv isolation in cell culture is critical for developing effective vaccines for ped prevention as well as performing various pedv research. however, the cell culture isolation of pedv has shown to be difficult and even the isolated virus may be unable to maintain infectivity upon further passages in cell culture (chen et al., ) . to date, there have only been two reports in more than two decades on the cultivation of the korean pedv strain that is genetically divergent from field pedvs (kweon et al., ; song et al., ) , while a number of pedv strains have been recently isolated in the us and successfully grown in cell culture for a year (chen et al., ; oka et al., ) , in the present study, we attempted to isolate pedv from various pedv-positive samples using vero cells. at this time, one highly virulent korean strain kor/knu- / has been successfully isolated and serially propagated in cell culture for over passages. we aimed to characterize the growth and titer of the pedv isolate knu- during the serial passages and the pathogenicity of the virus in suckling piglets. our in vivo assessment demonstrated that knu- is highly entero-pathogenic in piglets, exhibiting severe clinical symptoms as well as macroscopic and microscopic lesions typical for pedv infection. in addition, the complete genome or full-length s gene sequences of knu- were determined at selected passages to study the genetic stability and relationship. our data indicated that knu- isolate is relatively stable during the first passages in cell culture and is classified into subgroup g b that includes pedv strains responsible for recent severe outbreaks in korea and the us. vero cells (atcc ccl- ) were cultured in alpha minimum essential medium (␣-mem; invitrogen, carlsbad, ca) with % fetal bovine serum (fbs; invitrogen) and antibiotic-antimycotic solutions ( ×; invitrogen) and maintained at • c in a humidified % co incubator. seven small intestinal homogenates and stool specimens that tested positive by rt-pcr using an i-tge/ped detection kit (intron biotechnology, seongnam, south korea) were selected for virus isolation experiments. intestinal homogenates were prepared to % (wt/vol) suspensions with phosphate-buffered saline (pbs) using a magna lyser (roche diagnostics, mannheim, germany) by three repetitions of s at a speed of rpm. fecal samples were diluted with pbs to be % (wt/vol) suspensions. the suspensions were then vortexed and centrifuged for min at × g (hanil centrifuge fleta , incheon, south korea). the supernatant was filtered through a . -m-pore-size syringe filter (millipore, billerica, ma) and stored at − • c as an inoculum for virus isolation until use. virus isolation of pedv was attempted on vero cells as described previously with some modifications (hofmann and wyler, ) . briefly, prior to inoculation, inocula were prepared by adding trypsin (usb, cleveland, oh) to intestinal or fecal suspensions prepared above to give a final concentration of g/ml. confluent vero cells grown in -well plates were washed with pbs and inoculated with l of each inoculum containing trypsin. after incubating at • c for h, ml of virus growth medium [␣-mem supplemented with antibioticantimycotic solutions, . % tryptose phosphate broth (tpb; sigma, st. louis, mo), . % yeast extract (difco, detroit, mi), mm hepes (invitrogen), and g/ml of trypsin] was added. the inoculated cells were maintained at • c under % co and monitored daily for cytopathic effects (cpe). when % cpe appeared, inoculated cells were subjected to three rounds of freezing and thawing. the culture supernatants were then collected and centrifuged at × g for min. the clarified supernatants were aliquoted and stored at − • c as 'passage (p )' viral stocks until use. one hundred millimeter diameter tissue culture dishes were used for serial passages of the isolate. if no cpe was shown in inoculated cells for days, the plates were frozen and thawed three times, and the supernatants were harvested by centrifugation and inoculated on fresh vero cells for the next passage. if cpe and rt-pcr results were negative after blind passages, the virus isolation was considered negative. the pedv n protein-specific monoclonal antibody (mab) was obtained from choogang vaccine laboratory (cavac; daejeon, south korea). vero cells were infected with each passage knu- virus stock in the presence of trypsin as described above. the culture supernatants were collected at or h postinfection (hpi) at which a % cpe is commonly developed. for growth kinetics experiments, the supernatants were harvested from cells infected with each selected passage virus at different time points ( , , , , and hpi) and stored at − • c. virus titers were measured in -well plates by -fold serial dilution of the samples in triplicate per dilution to determine the quantity of viruses required to produce cpe in % of inoculated vero cells and calculated as tcid per ml using the reed-muench method (reed and muench, ) . the pedv titer was also determined by a plaque assay using vero cells and quantified as plaque-forming units (pfu) per ml. vero cells grown on microscope coverslips placed in -well tissue culture plates were mock infected or infected with pedv at a multiplicity of infection (moi) of . . the virus-infected cells were subsequently propagated until hpi, fixed with % paraformaldehyde for min at room temperature (rt) and permeabilized with . % triton x- in pbs at rt for min. the cells were blocked with % bovine serum albumin (bsa) in pbs for min at rt and then incubated with n-specific mab for h. after being washed five times in pbs, the cells were incubated for h at rt with a goat anti-mouse secondary antibody conjugated to alexa fluor (invitrogen), followed by counterstaining with , -diamidino- -phenylindole (dapi; sigma). the coverslips were mounted on microscope glass slides in mounting buffer and cell staining was visualized using a fluorescence leica dm il led microscope (leica, wetzlar, germany). viral rna was extracted from virus isolates or fecal samples prepared as described above using an i-tge/ped detection kit according to the manufacturer's protocol. quantitative real-time rt-pcr was performed using a one step sybr primescript rt-pcr kit (takara, otsu, japan) as described elsewhere (kim et al., ; sagong and lee, ) . the reaction took place using a thermal cycler dice real time system (takara) and the results were analyzed by the system software as described previously (sagong and lee, ) . eight - month-old guinea pigs (weighing - g) were randomly allocated into inoculated (n = ) and control (n = ) groups. six guinea pigs were immunized subcutaneously with . ml of binary ethylenimine (bei)-inactivated knu- -p virus in the presence of freund's complete adjuvant (sigma) and boosted once with a freshly prepared emulsion of the inactivated virus and freund's incomplete adjuvant (sigma) at a -week interval. two sham-inoculated guinea pigs were administered and boosted with cell culture media in the presence of the respective adjuvant. pre-immune sera were collected before starting the immunization and antisera were collected at weeks after the final boost. the presence of pedv-specific neutralizing antibodies in serum samples collected from guinea pigs in all groups was determined using a serum neutralization (sn) test in -well microtiter plates using pedv isolate knu- or vaccine strain sm - as previously described (oh et al., ) with minor modifications. briefly, vero cells were grown at × /well in -well tissue culture plates for day. the knu- -p virus stock was diluted in serum-free ␣-mem to make tcid in a l volume. the diluted virus was then mixed with l of -fold serial dilutions of individual inactivated sera in -well plates and incubated at • c for h. the mixture was inoculated into vero cells and incubated at • c for h. after removing the mixture, the cells were thoroughly rinsed times with pbs and maintained in virus growth medium at • c in a % co incubator for days. for the sn test using the vaccine strain, pedv strain sm - propagated in the absence of trypsin was diluted in serum-free ␣-mem to make pfu in a l volume. the diluted virus was then mixed with each serum sample in -well plates as described above and incubated at • c for h. subsequently, approximately × vero cells in l of ␣-mem containing % fbs were added to each well and the mixture was maintained at • c in a % co incubator for days. the neutralization titer was calculated as the reciprocal of the highest dilution of serum that inhibited virus-specific cpe in all of the duplicate wells. the in vivo swine studies described here were performed at the improah animal facility under the guidelines established by its institutional animal care and use committee. a total of suckling piglets of days of age were obtained from a commercial pig farm with no known prior ped outbreak or vaccination with pedv. all animals were determined to be free of antibodies to pedv as well as to transmissible gastroenteritis virus (tgev) and porcine reproductive and respiratory syndrome virus. pigs were randomly assigned to experimental groups housed in separated rooms: pedv-inoculated (n = ) and contact control (n = ) in room and sham-inoculated control (n = ) group in room . following a day acclimation period, only piglets in the pedv-inoculated group orally received a ml dose of tcid /ml of knu- -p virus. two piglets were exposed to the virus by direct contact with inoculated piglets in the same room. the sham-inoculated pigs were administered with cell culture media as a placebo. clinical signs of diarrhea and mortality were monitored daily for the duration of the study. stool samples from all groups were collected prior to inoculation and daily with inch, cotton-tipped swabs and subjected to rt-pcr using an i-tge/ped detection kit and real-time rt-pcr as described above. pedv-inoculated piglets were necropsied daily (days , , , and ) after challenge for post-mortem examinations throughout the study, whereas all pigs from the contact and control groups were euthanized at days post-challenge for post-mortem examinations. small intestinal tissue specimens (< mm thick) collected from each piglet were fixed with % formalin for h at rt and embedded in paraffin according to standard laboratory procedures. the formalin-fixed paraffin-embedded tissues were cut at - m thick on a microtome (leica), floated on a • c water bath containing distilled water, and transferred onto glass slides. the tissues were then deparaffinized in xylene for min and washed in decreasing concentrations of ethanol ( %, %, %, %, and %) for min each. the deparaffinized intestinal tissues sections were stained with hematoxylin and eosin (sigma) for histopathology or subjected to immunofluorescence assay using pedv n-specific mab as described above. the paraffin-embedded tissue sections were deparaffinized, treated with . m citrate buffer (ph . ) in a microwave oven for min, chilled at rt for min, and then incubated with . % hydrogen peroxide in dw for min to block endogenous peroxidase. after being washed three times in pbs, the sections were blocked with normal horse serum (vectastain abc kit; vector laboratories, burlingame, ca) and incubated h at rt with n-specific mab. after rinsing in pbs, the samples were reacted for min at rt with a horse anti-mouse secondary antibody (vectastain abc kit), incubated with avidin-biotin peroxidase complex for min (vectastain abc kit), and developed using the dab substrate kit (vector laboratories) according to the manufacturer's instructions. the slides were then counterstained with hematoxylin, dehydrated, cleared with xylene, and mounted on microscope glass slides in mounting buffer and tissue staining was visualized using a microscope. the complete genomic sequences of the original fecal sample as well as those of the p and p isolates were determined by nextgeneration sequencing (ngs) technology. total rna was extracted from the feces as well as p and p isolates using a rneasy mini kit (qiagen, hilden, germany) according to the manufacturer's instructions and used as a template to amplify cdna fragments as described elsewhere lee et al., b) . ten overlapping cdna fragments were generated to encompass the entire genome of each sample, pooled in equimolar amounts, and subjected to ngs using the ion torrent personal genome machine (pgm) sequencer system (life technologies, carlsbad, ca) and a v sequencing chip (life technologies) as described previously lee et al., b; rothberg et al., ) . the single-nucleotide variants (snvs) were analyzed using the clc genomic workbench version . (clc bio, cambridge, ma) and the individual ngs reads were assembled using the complete genome of pedv reference strain kor/knu- / (genbank accession no. kj ). the and ends of the genomes of the original feces and the p and p isolates were determined by rapid amplification of cdna ends (race) as described previously . the full-length genomic nucleotide sequences of the knu- -feces, knu- -p , and knu- -p were deposited in genbank under accession numbers kr , kr , and kr , respectively. the s glycoprotein gene sequences of the virus isolates were also determined by the traditional sanger method. two overlapping cdna fragments spanning the entire s gene of each isolate were rt-pcr amplified as described previously (lee et al., ) . the individual cdna amplicons were gel-purified, cloned into pgem-t easy (promega, madison, wi), and sequenced in both directions using two commercial vector-specific t and sp primers and the s gene-specific primers. in addition, the complete structural gene sequences of the virus isolate at selected passages (knu- -p , knu- -p , knu- -p , and knu- -p ) were determined by the sanger method as described above and deposited to the gen-bank database under their accession numbers shown in fig. . the sequences of fully sequenced s genes and complete genomes of pedv isolates were independently used in sequence alignments and phylogenetic analyses. the multiplesequencing alignments were generated with the clustalx . program (thompson et al., ) and the percentages of nucleotide sequence divergences were further assessed with the same software program. phylogenetic trees were constructed from the aligned nucleotide or amino acid sequences by using the neighborjoining method and subsequently subjected to bootstrap analysis with replicates in order to determine percentage reliability values on each internal node of the tree (saitou and nei, ) . all tree figures were produced using mega . software (tamura et al., ) . student's t test was used for all statistical analyses, and p-values of less than . were considered statistically significant. we attempted to isolate pedv from pcr-positive clinical samples, including feces and intestinal homogenates on vero cells. one pedv isolate designated knu- was successfully isolated from the feces of a naturally infected piglet from a commercial farm located in kyungpook province obtained on september , . the knu- virus was capable of producing distinct cpes typical for pedv infection, such as cell fusion, syncytium, and detachment, in infected vero cells from passage (p ). we then investigated whether the isolate can be efficiently cultivated and maintained in cell culture. thus, the isolated pedv strain knu- was further serially passaged in vero cells for a total of passages (p to p ). the time of cpe onset was at hpi and, accordingly, prominent cpe was observed within hpi in the first productive passages (p and p ). in the later passages, visible cpe appeared at hpi and became predominant by hpi. virus propagation was confirmed by detecting pedv antigens by ifa using a pedv n protein-specific mab. the distinct staining was distributed in the cytoplasm of typical syncytial cells. in contrast, no cpe and n-specific staining was evident in mock-inoculated vero cells. examples of cpe and corresponding ifa images in selected passages are shown in fig. . the level of viral genome in each selected passage was further assessed by real-time rt-pcr and the mean cycle threshold (ct) value was determined to be . , ranging from . (p ) to . (p ). the infectious titer of the isolate ranged from . to . tcid /ml up to p , whereas it was determined to be approximately tcid /ml in the later passages. the peak viral titer reached . tcid /ml (equivalent to . pfu/ml) or more since passage ( fig. a) . the growth kinetics study further showed that knu- replicated rapidly and efficiently in vero cells, reaching a maximum titer > tcid /ml by dpi (fig. b) . guinea pig antisera were collected before immunization (preimmune) and at weeks after the final boost and tested for their neutralizing activity against the isolate knu- or vaccine strain. as shown in fig. , the guinea pig antisera were highly effective in inhibiting knu- infection with mean neutralizing antibody (na) titers of : , whereas the antisera at relatively low dilution fully protect vero cells from sm - infection with mean na titers of : . in contrast, none of the pre-immune and nonimmunized sera showed neutralizing activity against either strain. taken together, our data indicated that the isolate knu- elicits potent antibody responses in immunized animals. however, the antisera strongly recognized the homologous field isolate, but inefficiently the heterologous pedv vaccine strain, suggesting the antigenic variations between the vaccine strain and field pedvs. four piglets were challenged orally with the knu- virus, while control animals were inoculated with cell culture media. two piglets were housed together with inoculated piglets in the same room for direct contact exposure. during the acclimation period, all piglets were active and had normal fecal consistency. pedv-challenged piglets exhibited clinical signs including lethargy and diarrheic feces by day post-inoculation (dpi) and experienced severe watery diarrhea with vomiting thereafter. pedv-associated mortality occurred in one of the inoculated pigs at dpi. contact piglets housed with the inoculated group exhibited diarrheic feces with vomiting by dpi and the progression of clinical signs was similar to that of the inoculated animals. furthermore, all inoculated and contact piglets were positive for pedv, as determined by rt-pcr, by or dpi and shed pedv in feces with mean ct values of . (range . - . ) and . (range . - . ), respectively. negative control pigs remained active with normal feces and continued to be undetectable for fecal shedding of pedv throughout the study period. one piglet died from pedv and was subsequently necropsied at dpi and the remaining inoculated piglets were randomly selected for necropsy thereafter. all contact and control animals were euthanized at the end of the study for postmortem assessments. all inoculated and naïve contact pigs macroscopically displayed typical ped-like lesions. the small intestine was dilated and accumulated with yellow fluid and its wall was thin and transparent, due to the villous atrophy (fig. , panel a) . the stomach was distended and filled with curdled and undigested milk. in contrast, the other intestinal organs appeared grossly normal. microscopic intestinal observations consistent with viral enteritis were developed in all inoculated and contact piglets, which included vacuolation of small intestinal enterocytes and shortening and fusion of small intestinal villi (fig. , panels b and c) . similar microscopic lesions were continuously present in the challenged and contact pigs through dpi. furthermore, both ifa and ihc staining revealed that the viral antigen was predominantly detected in the cytoplasm of epithelial cells on atrophied villi in all segments of the small intestines (fig. , panels d-f). these finding are comparable to those in pigs naturally or experimentally infected with virulent strains of pedv (debouck et isolates. pedv-specific cpes were observed daily and were photographed at hpi using an inverted microscope at a magnification of × (first panels). for immunostaining, infected cells were fixed at hpi and incubated with mab against the n protein, followed by alexa green-conjugated goat anti-mouse secondary antibody (second panels). the cells were then counterstained with dapi (third panels) and examined using a fluorescence microscope at × magnification. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) ). neither macroscopic nor microscopic intestinal lesions were observed in the negative control piglets during the experiment. entire genome sequence data of the original fecal sample (knu- -feces) and cell culture-passaged knu- -p and -p viruses were determined using the ngs technology. the full-length genome of kor/knu- / pedv was used as the initial reference for each ngs read and the individual complete genome sequences were successfully obtained by the assembly of respective ngs reads. the and ends of their genomes were also sequenced by race. all three genomes were , nucleotides (nt) in length, excluding the poly(a) tail and exhibited the genomic organization typical of all previously sequenced pedv strains, consisting of the -nt utr, the , -nt orf a/ b (nt to , for a and nt , to , for b), the -nt s gene (nt , to , ), the -nt orf (nt , to , ), the -nt e gene (nt , to , ) , the -nt membrane (m) gene (nt , to , ), the -nt n gene (nt , to , ), and the - fig. . virus-neutralizing antibody titers in the sera of guinea pigs inoculated with a pedv knu- isolate. guinea pigs were immunized twice with inactivated knu- -p virus by subcutaneous injection. blood samples were collected prior to immunization and at weeks after the second immunization and subjected to the virus neutralization assay using homologous (knu- ; solid circles) and heterologous (sm - ; solid diamonds) strains. neutralizing antibody titers for individual infected animals were spotted as a log scale. values are representative of the mean from three independent experiments in duplicate and error bars denote standard deviations. nt utr. the slippery heptamer sequence, tttaaac, followed by a stem-loop structure was present at the end of orf a in the pedv genome, which is a potential signal for a ribosomal frame shift during translation to generate the pp ab. the complete genome sequences of all three viruses were compared, and the results are summarized in table . compared to the knu- -feces, knu- -p showed one different nt at position , resulting in one amino acid (aa) change (leu to phe) in the s protein, while knu- -p gained two additional nt changes at positions , and , causing two aa mutations located in the s protein. the full-length s genes of the original feces and knu- viruses at selected passages (p , p , p , p , p , and p ) were also sequenced using the traditional sequencing method. the s protein sequences of knu- -feces, -p , and -p were completely identical to those determined by ngs. one nt change present in the s gene of knu- -p had been acquired since passage (knu- -p ). a total of three nt mutations, identified at passage compared to the feces, were sustained through passage (knu- - ) . in addition, compared to knu- -p , we were able to detect two independent mutations at positions , and , , resulting in amino acid changes in orf (asp to tyr) and e (pro to ser) of knu- -p , respectively, which were maintained at passage . altogether, our results revealed that the pedv isolate knu- is genetically stable during serial passages in cell culture. the entire genome sequences of pedvs determined in this study were further compared to those of other korean and non-korean pedv strains available in genbank, and the nucleotide homology and difference data are described in table . knu- isolates (feces, p , and p ) had the highest nucleotide identity ( . %) with the korean re-emergent strain kor-knu- and us strains, in and mn, showing to different nt at the genomic level. all three viruses were genetically distinct from the korean vaccine strains, sm - and dr- , and the prototype cv strain and exhibited relatively low nucleotide identity ranging from . % to . %. by alignment of the global pedv strains, a single-nucleotide insertion between nucleotides , and , has been previously identified in one chinese ah and three us strains, table nucleotide and amino acid changes of knu- during serial passages in cell culture. nucleotide amino acid position feces p p p p p p position feces p p p p p p utr ( ) -a -n d b nd --nd nd --nd nd --nd nd orf a ( , ) --nd nd --nd nd --nd nd --nd nd orf ab ( , ) --n d nd --nd nd --nd nd --nd nd s ( , ) a a a a c c c k k k k t t t c t t t t t t resulting in the shorter pp ab protein in length (chen et al., ) . however, none of the genomes of the three pedvs included such an insertion, and this result was further confirmed by the traditional sanger sequencing method (data not shown). in agreement with previous results (lee et al., ; lee and lee, ; lee et al., a,b) , the full-length s gene-based phylogenetic tree revealed that the global pedv strains were clearly defined into separate clusters, designated genogroup (g ; classical) and genogroup (g ; field epidemic). each genogroup can be further divided into subgroups a, b, a, and b (fig. a ). all original feces and passaged pedv viruses through passages were classified into subgroup b along with the recent korean field isolates, which were most closely clustered together with the emergent us strains in an adjacent clade with the same subgroup. subsequent phylogenetic analysis of the s region showed the same grouping structure as the s gene-based tree (data not shown). in addition, phylogenetic analysis based on the entire genome sequences demonstrated that strain knu- is grouped within the same cluster with the recent korean and us stains (fig. b ). in korea, three pedv strains, sm - , dr- , and chinju , were initially isolated almost two decades ago in korea. genetic and phylogenetic analyses revealed that sm - and dr- belong to the classical group , whereas chinju is classified into the field epidemic group . of these, only sm - and dr- isolates can be grown in cell culture and, accordingly, have been used as commercial vaccine seeds. since , we have been monitoring the genetic diversity of pedv prevalent in the field based on the s gene. our data demonstrated the existence of antigenic and genetic variations between vaccine and field strains, exhibiting a more than % amino acid difference in the s domain (lee et al., ; lee and lee, ; oh et al., ) . this may be one of the reasons for the incomplete efficacy of current vaccines in korea, suggesting that the isolation of field pedv is required for the development of a next-generation vaccine. although virus isolation in cell culture from clinical samples of naturally or experimentally infected pigs is fastidious, recent studies reported the successful isolation and propagation of several us original pedv strains using vero cells (chen et al., ; oka et al., ) . in this study, we initially sought to isolate pedv efficiently propagated in vitro from intestine homogenates and fecal samples of naturally infected piglets (field cases) and were able to obtain only isolate from feces in vero cell culture. the virus isolation rate, in the present study, was less than % and was relatively lower than that in recent us studies ranging from to % (chen et al., ; oka et al., ) . in previous studies, all isolated pedv strains originated from intestinal contents of naturally infected or experimentally inoculated pigs, suggesting that intestine samples may be a better source for virus isolation (chen et al., ; oka et al., ) . however, we failed to isolate pedv from intestine homogenates, which may be due to the number of intestinal contents (n = ) included in our study and be responsible for the low isolation rate. although pedv isolation might be affected by multiple factors, it appears to depend on the number of samples with good quality rather than the type of samples (intestinal contents or feces). further studies are needed to improve the isolation methodology or to determine the contributing factor(s) to enhance the success rate of pedv isolation in cell culture. the pedv isolate, knu- , was cytopathogenic in vero cells from passage and after passage , exhibited more severe and rapid cpe characterized by fusion of infected cells (syncytium or polykaryon formation). the initial viral infectious titers ranged from approximately to log tcid /ml and increased after several more passages reaching to log tcid /ml or more. these growth characteristics, including cytopathology, infectious titer, and growth kinetics were unchanged or even more efficient throughout the experiment ( passages), indicating that the isolate knu- is phenotypically stable during serial passages in vero cells. since the antibody response is a critical indicator to prove the cause of viral infection, we immunized guinea pigs twice with the inactivated isolate (knu- -p ) and determined whether the animals developed humoral immunity using an sn test. the guinea pig sera raised against the isolate contained high levels of na ( . log ), demonstrating the ability of knu- to elicit immune responses. on the other hand, the antisera showed a relatively weak neutralizing response (an almost -log reduction), when the heterologous vaccine strain sm - was used for an sn test. this weak neutralizing activity of the anti-knu- guinea pig sera against sm - was somewhat expected because of a high degree of genetic diversity between the s proteins of the vaccine strain and field isolates (lee et al., ; lee and lee, ; oh et al., ) . experimental oral inoculation of nursery pigs with pedv isolate knu- induced severe clinical disease typical of acute enteritis throughout the study, demonstrating that the isolate was highly enteropathogenic in neonatal piglets. onset of clinical signs including lethargy and watery diarrhea in inoculated pigs was found as early as dpi. the pedv genome was detected in feces in % of challenged pigs on dpi and virus quantities from fecal shedding were maintained thereafter in all challenged pigs throughout the study period, leading to the source for direct transmission of virus to other animals. this result is inconsistent with a previous study using a us pedv strain that demonstrated virus fecal shedding on dpi in only one-fourth of the challenged pigs (madson fig. . phylogenetic analyses based on the nucleotide sequences of the spike genes (a) and the full-length genomes (b) of pedv strains. a putative similar region of the spike protein and the complete genome sequence of tgev was included as an outgroup in each panel. multiple-sequencing alignments were performed using clustalx program and the phylogenetic tree was constructed from the aligned nucleotide sequences by using the neighbor-joining method. numbers at each branch represent bootstrap values greater than % of replicates. names of the strains, countries and years of isolation, genbank accession numbers, and genogroups and subgroups proposed in this study are shown. the pedv isolates identified in this study are indicated by solid circles. scale bars indicate nucleotide substitutions per site. et al., ) . the difference between the current and previous studies is the age of the pigs: -week-old neonatal pigs and -week-old weaned pigs used in the present and previous experiments, respectively. therefore, younger piglets in this study were more sensitive to pedv infection shedding virus in feces earlier than older pigs, probably due to an age-dependent disease severity as previously described (shibata et al., ) . ihc and ifa revealed that viral antigen in villous enterocytes were observed at dpi in all segments of the small intestine of inoculated piglets. the onset of clinical signs and viral fecal shedding and the detection time of viral antigen in the target tissue were similar to recent independent reports using different us pedv strains jung et al., ) . two non-challenged piglets were included in this study for direct contact to inoculated piglets in the same space. all contact piglets displayed ped-like symptoms within h after the onset of clinical signs in inoculated piglets. the presence of pedv in feces and infected tissues was further verified in contact piglets, showing % morbidity in our study. mortality averages % in suckling piglets up to week of age, often approaching % in -to -dayold piglets, and decreases to % thereafter (saif et al., ) . in our study, mortality was observed only in one out of four inoculated piglets, resulting in % mortality in the current study involving -day-old piglets. for reproducible challenge studies in vivo using the isolate in the future, a neonatal swine bioassay will be needed to determine either the median pig diarrhea dose or lethal dose as a standardized dose and this aspect is currently under investigation. whole-genome sequences of korean pedv strains (knu- -feces, p , and p ) were determined using ngs approaches coupled with race experiments. regions covering the structural genes were also sequenced at the selected passages by the sanger method for confirmation. compared to the original feces (knu- -feces), only one nt difference at position , was identified for the first passages (knu- -p ) in cell culture, which led to a non-synonymous mutation at the corresponding position of the s protein. this nt change was initially found at passage and further maintained through passage . however, we were unable to investigate whether this mutation had been acquired at the beginning of the vero cell culture since infectious virus was not obtained during the first two passages. interestingly, the identical c t mutation at the whole-genome level (l f at the aa level of s) has been previously reported in a us pedv isolate isu- e during cell culture passage (chen et al., ) , suggesting its potential importance for adaption of the field virus to growth in vitro. at passage , two more nt differences at positions , and , of the genome (a c and g c) were detected when compared to knu- -feces. all of the changes acquired in knu- -p led to non-synonymous mutations at the respective positions and of the s protein (k t and e q). these findings were similar to recent data reported by chen et al. ( ) that two us isolates individually gained the mutations located in orf b, s, and e through passage in cell culture. however, no nucleotide changes were identified in orf b and e for the first passages in vero cells. the mutations in the s protein were persistent for passages in cell culture. celladapted pedv vaccine strains, sm - and dr- , are known to contain a -nt deletion spanned from the end of s to the start of orf and a -nt deletion in the middle of orf . thus, we further sequenced structural genes of knu- -p and -p and identified two additional nucleotide changes (g t and c t) acquiring non-synonymous amino acid mutations in orf and e, respectively. except for those nucleotide substitutions, the isolate had no extra change, including such large deletions in structural genes through passage , indicating the genetic stability of knu- during serial passages in vero cells. sequence comparisons with other pedv strains showed that knu- isolates are genetically most similar to recent korean and us epidemic strains with . - . % identities, but most distinctly related to classical strains, cv , sm - , and dr- , with . - . % identities at the genomic level. all phylogenetic analyses based on the complete genome, the full-length s gene, and the s portion formed the similar tree structure, revealing that the korean knu- strains are clustered together with the re-emergent korean strains in subgroup b that also includes emergent us strains and - field epidemic chinese strains. in conclusion, we isolated and serially propagated pedv in cell culture that is phenotypically and genotypically identical to field strains responsible for the recent severe ped outbreaks in korea. to our knowledge, this is the first report describing the isolation and in vitro and in vivo characterization of korean pedv associated with the field epidemic. with the availability of the korean isolate, we are now able to spur the development of new effective and safe vaccines for ped prevention. indeed, our pedv isolate has been used for the development of an inactivated vaccine that is currently being evaluated under experimental and field conditions. furthermore, we are continuing to passage the isolate in vero cells to develop a live attenuated vaccine that generally prove to provide a more efficient protective immunity than killed viral vaccines. for this purpose, pathogenic and molecular characterization of the isolates at selected passages will be assessed to determine their phenotypes in pigs and to identify the genetic changes involved in pedv attenuation. molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in china isolation and characterization of porcine epidemic diarrhea viruses associated with the disease outbreak among swine in the united states experimental infection of pigs with a new porcine enteric coronavirus, cv the pathogenesis of an enteric infection in pigs, experimentally 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, and several coronaviruses porcine epidemic diarrhoea virus as a cause of persistent diarrhoea in a herd of breeding and finishing pigs chinese-like strain of porcine epidemic diarrhea virus a simple method for estimating fifty percent endpoints an integrated semiconductor device enabling non-optical genome sequencing porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-b production by inhibiting irf activation in immortalized porcine alveolar macrophages coronaviruses the neighbor-joining method: a new method for reconstructing phylogenetic trees isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages differentiation of a vero cell adapted porcine epidemic diarrhea virus from korean field strains by restriction fragment length polymorphism analysis of orf emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan mega : molecular evolutionary genetics analysis (mega) software version . the clustal x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools distinct characteristics and complex evolution of pedv strains we gratefully thank gun-seok park and jae-ho shin from kyungpook national university for their help in ngs analysis. this research was supported by basic science research program through the national research foundation of korea (nrf) funded by the ministry of education, science and technology(nrf- r a a ). key: cord- -cdvnqfjz authors: castilla, v.; mersich, s. e.; candurra, n. a.; damonte, e. b. title: the entry of junin virus into vero cells date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: cdvnqfjz the entry mechanism of junin virus (jv) into vero cells was studied analyzing the effect of lysosomotropic compounds and acid ph on jv infection. ammonium chloride, amantadine, chlorpheniramine and procaine inhibited jv production. the action of ammonium chloride was exerted at early times of infection. virus internalization was inhibited and viral protein expression was not detected. when the extracellular medium was buffered at low ph, the ammonium chloride induced block on jv infection was overcome. furthermore, jv was able to induce fusion of infected cells at ph . leading to polykaryoctye formation. taken together, these results demonstrate that jv entry occurs through an endocytic mechanism requiring a low ph dependent membrane fusion. junin virus (jv) is a member of the arenaviridae family of enveloped rna viruses. virions contain a genome composed of two segments of single-stranded rna and two major structural proteins, the nucleocapsid protein (np, mw - kda) and an external glycoprotein (gp , mw kda) [ ] . other minor proteins have also been described in purified virions or in infected cells [ , , ] . the main biological properties of jv are its ability to establish persistently infected cultures in-vitro and to produce chronic infections in the field mouse calomys musculinus [ ] . in humans, jv induces argentine haemorrhagic fever, an endemoepidemic disease with hematologic and neurologic signs, which mainly affects male rural workers [- ] . although some features of arenavirus replication have been described, the early events of the multiplication cycle remain poorly characterized. main interest has been focussed on the knowledge of viral rna transcription and replication, the characterization of the ambisense genome strategy and the study of protein structure and expression [- , ] . little is known about the process of virus attachment and entry into the cell. a brief report has recently suggested that the replication of the arenaviruses pichinde, mopeia and lassa was sensitive to some lysosomotropic compounds [ ] . two distinct pathways operate for the entry of enveloped viruses into animal cells: some viruses penetrate by direct fusion of the viral envelope with the plasma membrane while other viruses are taken up by an endocytic mechanism [ ] . the endocytosed virions travel to intracellular compartments where acidic conditions facilitate fusion between the viral envelope and the vesicle membrane releasing the nucleocapsid into the cytoplasm. thus, lysosomotropic agents such as weak bases and carboxylic ionophores that elevate the ph of acidic organelles, interfere with the virion uncoating and inhibit the replication [ , , ] . the uncharged form of any weak base has been proposed to enter the acidic intracellular compartments very efficiently, raising the ph as it becomes protonated and inhibiting hydrolytic enzymes [ ] . in this paper, we report studies on jv penetration into vero cells analyzing the effect of acidic ph and lysosomotropic bases on virus entry. monolayers of vero cells were grown in minimum essential medium (mem) supplemented with % calf serum and gentamycin. the iv strain of jv [ ] was used. stock solutions of amantadine (specia), ammonium chloride (fisher company), chlorpheniramine (schering-plough) and procaine (schering-plough) at concentrations of , , and mm, respectively, were prepared in culture medium and sterilized by filtration. ammonium chloride ( mm) was added to vero cells h before infection with jv or at , t, , or h post-infection (p.i.). in all cases supernatant cultures were harvested at h p.i. and extracellular virus yields were determined by plaque assay. vero cells were infected with jv (virus inoculum: × pfu) in the presence or in the absence of mm ammonium chloride. after adsorption for , , , , and rain at °c, infected cells were washed twice with cold phosphate buffer saline (pbs) and disrupted by freezing and thawing. the amount of infectious bound virus was then determined by plaque assay. cultures of vero cells were infected with jv at a moi of . after rain adsorption at °c two washes were done to remove excess inoculum and then cultures were warmed to °c for various times in the presence or absence of mm ammonium chloride. cultures were subsequently treated with proteinase k ( . mg/ml) in pbs for min at °c in order to remove external virus. protease treatment was stopped by the addition of mm phenyl methyl sulfonyl fluoride (pmsf), ~ bovine serum albumin (bsa) in pbs. cells were centrifuged min at g and the resultant pellet was washed and resuspended in mem. internalized virus was measured by an infectious center assay on vero cells. vero cells grown in coverslips were infected with jv (moi ) and mm ammonium chloride was added to the culture medium after adsorption. at h p.i. supernatants were removed. monolayers were washed with cold pbs, fixed in methanol and stained for cytoplasmic immunofluorescence with anti-jv purified immunoglobulins as previously described [ ] . veto cell monolayers in -wetl plates were adsorbed for h at °c with jv in mem containing . ~/o bsa, mm hepes, ph . . in a set of cultures cells were washed twice with pbs and then warmed to °c by the addition of pre-warmed medium buffered with mm hepes and mm - -piperazinediethene-sulfonic acid (pipes) at ph . , . , . , . , . and . , in the presence or in the absence of mm ammonium chloride. after h of incubation at °c, cells were washed twice with pbs and mem containing . ~ methylcellulose, ph . , was added. in other set of cultures, after adsorption cells were washed with pbs and incubated at °c for rain in mem buffered at different ph as indicated above. then, cells were washed with pbs and mem at ph . containing or not ram ammonium chloride was added. after h of incubation cultures were overlaid with methylcetlulose as above. in both set of cultures plaques were counted after days of incubation and percent of inhibition was calculated. vero cells were infected with jv at a moi of . at h after infection, medium was removed and cells were incubated in mem buffered at ph . or . as indicated above. then, monolayers were stained with giemsa and syncytia containing more than nuclei were counted. to determine the time at which lysosomotropic agents caused their inhibitory action, we next examined the effect of the time of addition of mm ammonium chloride on extracellular virus yields. a nearly complete inhibition of viral multiplication was observed when the compound was added one hour before or simultaneously with virus infection and maintained during all the period of incubation (fig. ) . when time of drug addition was delayed, virus production progressively increased, indicating that ammonium chloride inhibited an early step of the replicative cycle. in fact, no significant differences were found when the drug was added between and h p.i. since maximum inhibition in jv multiplication was obtained when the drug was present during the first hour after infection, we examined its effect on the early stages of replication. as shown in table , ammonium chloride had no effect on jv adsorption since the titers of virus adsorbed at °c in treated and untreated cells did not differ significantly. in both cases, virus adsorption occurred rapidly and the percentage of adsorbed virus was approximately %. to investigate whether ammonium chloride affects jv internalization, virus adsorbed cells at °c were warmed to ~c for various intervals in presence or absence of the compound and internalized virus was determined by infectious center assay. the amount of jv internalized virus in untreated cells sharply increased after min of incubation at °c and the uptake was apparently complete after . h (fig. ) . in the presence of ammonium chloride there was no virus internalization and a progressive decrease is observed in the number of infectious centers. the relatively small difference between the control and treated cells at the min time point might reflect the fact that part of the adsorbed virus was not removed by the proteinase k treatment. the action of weak bases on jv replication was confirmed by measuring viral protein production in infected cells by an immunofluorescence assay. the inhibition in the number of fluorescent cells in the presence of mm ammonium chloride was . % (fig. ) . the other lysosomotropic compounds also inhibited viral antigen expression (data not shown). in an effort to demonstrate that the entry of jv into vero cells is an acid ph-dependent process, we have finally studied the effect of different ph values on the internalization of jv particles that have been prebound to vero cells. in the absence of ammonium chloride, infectivity of jv was not affected at the ph range assayed ( . - . ) (data not shown). a high degree of inhibition was observed when cultures were incubated in medium containing ammonium chloride at ph . , . and . during h, while there was only . and . % inhibition at ph .t and . , respectively (fig. ) . at more acidic ph values the action of ammonium chloride was almost totally abolished. to assess that the acid ph is modifying the mechanism of viral entry and is not just neutralizing the ammonium chloride, jv bound cells were briefly treated with medium buffered at different ph for min and then incubated for h in neutral medium containing or not ammonium chloride. this treatment prevents endocytosis by inhibiting release into the cytoplasm of virus entered in endocytic vacuoles and allows virus entry only by fusion at the plasma membrane [ ] . under these experimental conditions, ammonium chloride inhibition was partially reversed at ph . (fig. ). to reinforce that the membrane fusion activity of junin virus is expressed at low ph, the formation of jv-induced syncytia in infected vero cells incubated in medium at ph . or . was quantitated. cell fusion was observed at h p.i. but only at ph . , there being more than nuclei in the syncytia whereas no polykaryon formation was seen at neutral ph (fig. ). these studies present evidence that junin virus entry occurs via a ph dependent endocytic mechanism mediated by a glycoprotein fusogenic activity. acidotropic bases are known to disrupt endosomal functions when their uncharged forms enter endosomes and lysosomes, become protonated, raise the ph and inhibit the hydrolytic enzymes [ ] . we have demonstrated here (figs. , ) . the main action of a m m o n i u m chloride on jv replication was exerted at an early step in the multiplication cycle (fig. ) . however, virus attachment which occurred at °c was not inhibited as it is shown in table . virions b o u n d to cells at °c are compelled to internalize when the temperature of incubation is raised to °c. when jv internalization, the next stage of the viral cycle, was studied a significant inhibition was observed during the first v. castilla et al. hour after adsorption. in the presence of ammonium chloride an increasing reduction in the number of proteinase k-resistant infectious centers was detected (fig. .) it might be due to the lysosomal degradation of endocytosed virions unable to fuse with endosomal membranes, as seen for other enveloped viruses. these data were indicative of a process of low ph-dependent endosomal fusion for jv uptake. in fact, a bypass of the ammonium chloride block of jv infection was achieved when the extracellular medium was at a ph below . (fig. ) , suggesting that the acidic conditions would probably trigger direct fusion of virus envelope with the cell membrane. the location of penetration is determined by the ph threshold of fusion activity [ ] . for some viruses such as semliki forest virus, fusion occurs at a ph . in early endosomes, whereas influenza virus x- requires ph . and fuses in the late endosomes [ , , ] . for jv entry, fusion seems to occur in endosomes where the ph is . or lower. the acid environment may be responsible for structural changes in jv external proteins allowing membrane fusion and facilitating viral uncoating as described for influenza virus [ ], rubella virus [ ] , west nile virus [ ] and tick-borne encephalitis virus [ ] . it is possible to induce fusion in some model systems by mimicking the low ph intracellular conditions. in particular, mann et al. [ - have shown that sindbis virus infected cells express a fusion function after treatment at acid ph. we demonstrated that junin virus can mediate cell fusion at ph . producing polykaryocytes in which over % of the cells in the monolayer participate (fig. ) . thus, this result offers indirect support for the conclusion that the route of jv entry is ph-dependent in vero cells. major expression of gp , the main external envelope glycoprotein [ ] , was observed in the surface of jv infected cells at h p.i., by immunofluorescence assay (data not shown). thus, gp might be responsible for jv-induced membrane fusion in the endosome or in the cell surface. this proposal is also supported by results obtained with c , a host-range mutant of jv, with an alteration in gp detected by peptide mapping and a blockade in adsorption-penetration pathway [ , ] . in conclusion, our data demonstrate for the first time that jv enters the cell by a receptor mediated endocytic pathway and that low ph is neccesary for viral internalization through a fusing activity. further experiments are in progress to determine the precise role of gp in virus entry and the nature of the conformational changes at low ph leading to membrane fusion. the entry of african swine fever virus into vero cells arenavirus gene structure and organization protein structure and expression among arenaviruses antigenic relationships among attenuated and pathogenic strains of junin virus coto ce (t ) a comparison of junin virus strains growth characteristics, cytopathogenicity and viral polypeptides polypeptide synthesis in junin virus infected bhk- cells membrane fusion activity of the influenza virus hemagglutinin early events in arenavirus replication are sensitive to lysosomotropic compounds the uncoating and infection of the flavivirus west nile on interaction with cells: effects of ph and ammonium chloride junin virus structure epitope model of tick-borne encephalitis virus envelope glycoprotein e: analysis of structural properties, role of carbohydrate side chain and conformational changes occurring at acidic ph kinetics of endosome acidification detected by mutant and wild type semliki forest virus association between the ph-dependent conformational change of west-nile flavivirus e protein and virus-mediated membrane fusion attenuation of murine coronavirus infection by ammonium chloride virus entry into animal cells polykaryocyte formation mediated by sindbis virus glycoproteins ph-dependent solubility shift of rubella virus capsid proteins weak bases and ionophores rapidly and reversibly raise the ph of endocytic vesicles in cultured mouse fibroblast lectin affinity of junin virus glycoproteins genetic organization of junin virus, the etiological agent of argentine hemorrhagic fever reduced virulence ofa junin virus mutant is associated with restricted multiplication in murine cells damonte eb (t ) a mouse attenuated mutant of junin virus with an altered glycoprotein the entry of junin virus into vero cells the effects of oligosaccharide trimming inhibitors on glycoprotein expression and infectivity of junin virus helenius a (t ) protein mediated membrane fusion fusion of influenza virions in an intracellular acidic compartment measured by fluorescence dequenching membrane fusion process of semliki forest virus: low ph-induced rearrangement in spike protein quaternary structure precedes virus penetration into cells argentine hemorrhagic fever we thank s. coronato for her technical assistance. this work was supported by grants from the consejo nacional de investigaciones cientificas y t cnicas (conicet) and universidad de buenos aires. e.b. damonte is member of the research career from conicet. key: cord- -jfjrk gy authors: fang, shouguo; chen, bo; tay, felicia p.l.; ng, beng sern; liu, ding xing title: an arginine-to-proline mutation in a domain with undefined functions within the helicase protein (nsp ) is lethal to the coronavirus infectious bronchitis virus in cultured cells date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: jfjrk gy genetic manipulation of the rna genomes by reverse genetics is a powerful tool to study the molecular biology and pathogenesis of rna viruses. during construction of an infectious clone from a vero cell-adapted coronavirus infectious bronchitis virus (ibv), we found that a g–c point mutation at nucleotide position , causing arg-to-pro mutation at amino acid position of the helicase protein, is lethal to the infectivity of ibv on vero cells. when the in vitro-synthesized full-length transcripts containing this mutation were introduced into vero cells, no infectious virus was rescued. upon correction of the mutation, infectious virus was recovered. further characterization of the in vitro-synthesized full-length transcripts containing the g c mutation demonstrated that this mutation may block the transcription of subgenomic rnas. substitution mutation of the arg residue to a positively charged amino acid lys affected neither the infectivity of the in vitro-synthesized transcripts nor the growth properties of the rescued virus. however, mutation of the arg residue to leu, a conserved residue in other coronaviruses at the same position, reduced the recovery rate of the in vitro-synthesized transcripts. the recovered mutant virus showed much smaller-sized plaques. on the contrary, a g–c and a g–a point mutations at nucleotide positions and , respectively, causing glu–gln and gly–glu mutations in or near the catalytic centers of the papain-like (nsp ) and c-like (nsp ) proteinases, did not show detectable detrimental effect on the rescue of infectious viruses and the infectivity of the rescued viruses. coronaviruses cause severe diseases affecting human and other animal species. in , a novel coronavirus (sars-cov) was identified as the causative agent of severe acute respiratory syndrome (sars) (marra et al., ; rota et al., ) . the potential risk to public health posed by sars-cov and other coronaviruses and the lack of specific antiviral agents and vaccines have triggered a global research effort to characterize this family of viruses at the molecular and cellular levels. major advances in studies of the biological functions of individual viral proteins and replication mechanisms are currently being made by genetic manipulation of coronaviral genomes using reverse genetics and targeted recombination approaches (almazan et al., ; casais et al., casais et al., , casais et al., , coley et al., ; hodgson et al., ; koetzner et al., ; masters and rottier, ; sanchez et al., ; youn et al., a youn et al., , b yount et al., yount et al., , yount et al., , yount et al., , . avian infectious bronchitis virus (ibv), a group coronavirus, causes an acute and contagious disease in chickens with a significant impact on the poultry industry worldwide. ibv contains a . kb single-stranded, positive-sense rna genome. in the virus-infected cells, six mrna species, including the genome-length mrna and five subgenomic mrnas (mrna - ), are produced by a discontinuous rna transcription mechanism. each mrna species possesses a nucleotides leader sequence derived from the ′ end of the virology ( ) - www.elsevier.com/locate/yviro genome (boursnell et al., ) . subgenomic mrnas , , , and encode the four structural proteins, i.e., spike glycoprotein (s), envelope protein (e), membrane protein (m), and nucleocapsid protein (n). the ′ two-third region of mrna comprises two large orfs, a and b, and encodes two polyproteins. the two polyproteins are proteolytically cleaved by two virus-encoded proteinases, the papain-like and c-like proteinases, into functional proteins (nsp -nsp ) liu, a, b; lim et al., ; liu et al., liu et al., , liu et al., , ng and liu, xu et al., ) . compared to other coronaviruses, nsp is absent in ibv but nsp is considerably larger liu, a, b; liu et al., ) . in general, ibv shares close similarities in the genome organization, gene expression, and rna replication with other coronaviruses but is non-infectious to human. these properties make ibv an attractive model system for studying the biology and pathogenesis of coronavirus. in construction of an infectious ibv clone by in vitro assembly of five cloned rt-pcr fragments from a vero celladapted ibv beaudette strain, a g-c (g c) point mutation at nucleotide position , was found to be lethal to the infectivity of ibv on vero cells. no infectious virus could be rescued from vero cells electroporated with in vitro-synthesized full-length transcripts containing this mutation. as this mutation causes arg -pro mutation in a domain with unknown function within the helicase protein (nsp ), it implies that this region might play certain roles in the functionality of the helicase protein. on the contrary, a g-c (g c) and a g-a (g ) point mutations at nucleotide positions and , respectively, causing glu-gln and gly-glu mutations in or near the catalytic centers of the papain-like (nsp ) and c-like (nsp ) proteinases, did not impair the infectivity of the in vitrosynthesized transcripts containing these mutations. further characterization of the in vitro-synthesized full-length transcripts containing the g c mutation demonstrated that this mutation blocks the transcription of subgenomic rnas. substitution mutation of the arg residue to a positively charged amino acid (lys) affected neither the infectivity of the in vitro-synthesized transcripts nor the growth properties of the rescued virus. however, mutation of the arg residue to a leu, which is conserved in most of other coronaviruses at the same position, reduced the viral recovery rate of the in vitro- fig. . in vitro assembly of full-length cdna clone derived from a vero cell-adapted ibv beaudette strain. (a) diagram of the genome organization of ibv. the regions coding for the replicase polyproteins, the structural proteins s, e, m, and n, the accessory proteins a, b, a, and b, and the ′-and ′-utr are shown. also shown are the regions of the five rt-pcr fragments, the t promoter at the ′-end of fragment a, and the as at the ′-end of fragment e. (b) preparation of the five cdna fragments, assembly of the five fragments into a full-length cdna clone, and in vitro transcription of the full-length transcripts. the five cdna fragments covering ibv sequences from nucleotides - (lane ), - (lane ), - , (lane ), , - , (lane ) , and , - , (lane ), respectively, were obtained by digestion of corresponding plasmid dna with either bsmbi or bsai, purified from agarose gel, and analyzed on a . % agarose gel (lanes - ). equal amounts of the purified fragments were ligated using t dna ligase (lane ) and analyzed on a . % agarose gel. the in vitro assembled full-length cdna was used as templates for generation of the full-length in vitro transcripts, which were analyzed on a . % agarose gel (lane ). lanes , , and show dna markers, and numbers on the left indicate nucleotides in kilobases. synthesized full-length transcripts. the mutant virus showed much smaller-sized plaques. this study reveals the essential role of a domain with previously unassigned functions within the helicase protein in coronavirus replication. construction of a full-length cdna clone derived from a vero cell-adapted ibv beaudette strain by in vitro assembly of five rt-pcr fragments to construct a full-length ibv clone, five fragments (a to e) spanning the entire ibv genome were obtained by rt-pcr of total rna extracted from vero cells infected with a vero cell-adapted ibv beaudette strain (p ) (shen et al., (shen et al., , fang et al., ) . to facilitate the assembly of the full-length cdna in vitro, restriction sites for either bsmbi or bsai were introduced into both the ′ and ′ ends of the fragments (fig. a) . in fragment a, a -nucleotide sequence corresponding to the t rna promoter (table ) was inserted into the ′ end of the ibv genome to facilitate in vitro transcription using the t polymerase (fig. a) . the primers used to amplify these fragments are listed in table . the pcr products were purified from agarose gel and cloned into either pcr-xl-topo (invitrogen) or pgem-t easy (promage) vectors. for the convenience of digestion using the restriction enzyme bsmbi, the nhei-and ecori-digested fragment a was subcloned into pkt which contains a bsmbi site bp upstream of the t promoter sequence (fig. a) . two to three independent clones for each construct were selected for sequencing. the complete sequences of the five fragments, determined by automated nucleotide sequencing, are summarized in table . the five fragments were then prepared by digestion of the corresponding constructs with either bsmbi or bsai and purified (fig. b, lanes - ) . the full-length clone was made by ligation of the purified fragments in vitro (fig. b, lane ) and used as the template for in vitro transcription. the fulllength in vitro-synthesized transcripts were generated using the mmessage mmachine t kit (ambion, austin, tex) (fig. b , lane ). as coronavirus n gene transcripts were shown to enhance the recovery of the rescued virus from the in vitrosynthesized full-length transcripts (casais et al., ; youn et al., a youn et al., , b yount et al., yount et al., , , the n transcripts were generated from a linearized pkt -ibvn construct containing ibv n gene and the ′-utr region. the full-length transcripts together with the n transcripts were introduced into vero cells by electroporation. however, it was consistently observed that no infectious virus could be recovered from cells transfected with the full-length transcripts together with the n transcripts. sequencing comparison of the five fragments with the vero cell-adapted ibv strain (p , accession no. dq ) shows nucleotide changes at positions (table ). among them, a unique mutations found only in the cloned fragments. b same as the published sequences but different from p . caused unique amino acid changes (table ) . to assess the deleterious effects of these mutations on the infectivity of the full-length clone, correction of some of the mutations was carried out by site-directed mutagenesis. three point mutations, g c, g a, and g c, were chosen based on the fact that they are located either in or near the catalytic centers of the two viral proteinases or in a region of the rna helicase protein with undefined functions. four full-length cdna clones either with correction of the mutations at all three positions (ribv) or combination of two positions (e.g., g c containing g-c mutation at nucleotide position but without mutations at the other two positions) were constructed. rna transcripts generated from the four full-length cdna clones were introduced into vero cells together with the n transcripts by electroporation. at days post-electroporation, a typical cpe of the vero cell-adapted ibv, the formation of giant syncytial cells (fang et al., ) , was observed in cells transfected with transcripts generated from cdna clones ribv, g c, and g a. cpe was extended to almost the whole monolayers at days post-electroporation ( fig. a) . no cpe was observed in cells transfected with transcripts generated from clone g c (fig. a) . rt-pcr analysis of the subgenomic mrna was performed to confirm if cpe observed is caused by the replication of ibv. sequencing of the rt-pcr fragment generated from cells transfected with ribv showed correct sequence in the leader/body junction region of the subgenomic mrna (fig. b ). further sequencing of the rt-pcr fragments covering regions with unique amino acid mutations confirmed the recovery of ibv (ribv) from the in vitro-synthesized fulllength transcripts. compared to the parent ibv strains, ribv contains amino acid mutations (table ). to test if these mutations may affect the growth properties and genetic stability of the rescued virus, ribv was propagated on vero cells for passages, and the plaque sizes and growth kinetics were determined and compared with wild type ibv p . in cells infected with ribv, the average plague size is . ± . mm, which is slightly smaller than the average plaque size of . ± . mm in cells infected with wild type ibv (fig. a) . analysis of the growth curves demonstrated that ribv exhibited very similar growth properties as the wild type virus (fig. a) . further characterization of ribv was subsequently carried out by analysis of viral rnas and structural proteins. northern blot and western blot analyses showed the detection of very similar amounts of viral rnas (fig. b ), and s, n, and m proteins ( fig. c ) in cells infected with wild type and ribv, respectively, at h post-infection. when probing with anti-n protein antibodies, other species migrating faster than the fulllength products on sds-page were also observed ( fig. c ). they may represent premature termination and cleavage products of n protein (unpublished observation). it was also noted that variable amounts of these species were detected in cells infected with wild type and ribv (fig. c ). the significance of these variations is unclear at the moment. taken together, these results confirm that ribv is stable and possesses very similar growth properties as wild type ibv. as no infectious virus was recovered from cells transfected with g c mutant transcripts, rt-pcr amplification of the negative strand rna was performed to check if rna replication occurred in these transfected cells. total rna was extracted from vero cells transfected with wild type and g c mutant transcripts at and h post-electroporation, respectively. reverse transcription was performed by using equal amount of rna and the sense-primer ibv -f ( ′- , gct-tatccactagtacatc , - ′), and pcr was carried out by using the sense-primer ibv -f and the antisense-primer ibv -r ( ′- , cttctcgcacttctgcacta-gca , - ′). if replication of viral rna occurred, a bp pcr fragment would be expected. as shown in fig. a , rt-pcr fragments amplified from negative strand rna templates were obtained from cells transfected with both wild type (lanes and ) and the mutant transcripts (lanes and ). sequencing of the pcr fragments confirmed that they represent the correct sequences. as a negative control, the in vitro-synthesized transcripts were mixed with total rna extracted from normal vero cells and were used as a template for rt-pcr. no rt-pcr fragment was detected (fig. a, lane ) , confirming that the detection of negative strand rna from cells transfected with mutant transcripts is due to the replication of viral rna. further quantitation of the negative strand rna transcription in cells transfected with wild type and g c mutant transcripts was carried out by real-time pcr. at and h post-electroporation, transcription of the negative rna in cells electroporated with wild type transcripts was . -and . -fold, respectively, higher than that in cells electroporated with the g c mutant transcripts. these results confirm that transcription of the negative strand rna has taken place in cells transfected with the mutant transcripts, but with much lower efficiency. fig. . analysis of the growth properties of wild type (p ) and ribv. (a) plague sizes and one-step growth curves of wild type and ribv. monolayers of vero cells on a -well plate were infected with μl of -fold diluted virus stock and cultured in the presence of . % carboxymethy cellulose at °c for days. the cells were fixed and stained with . % toluidine. to determine the one-step growth curves of wild type and ribv, vero cells were infected with the viruses and harvested at , , , , , , and h post-inoculation, respectively. viral stocks were prepared by freezing/thawing of the cells three times, and tcid of each viral stock was determined by infecting five wells of vero cells on -well plates in triplicate with -fold serial dilution of each viral stock. error bar shows standard error of the mean. (b) northern blot analysis of the genomic and subgenomic rnas in cells infected with wild type and ribv. ten micrograms of total rna extracted from vero cells infected with wild type and ribv, respectively, was separated on % agarose gel and transferred to a hybond n+ membrane. viral rnas were probed with a dig-labeled dna probe corresponding to the -end nucleotides of the ibv genome. total rna extracted from mock-infected cells was included as negative control. numbers on the left indicate nucleotides in kilobase, and numbers on the right indicate the genomic and subgenomic rna species of ibv. (c) western blot analysis of viral protein expression in cells infected with wild type and ribv. vero cells infected with wild type (lane ) and ribv (lane ) were harvested, lysates prepared and separated on sds- % polyacrylamide gel. the expression of s, n, and m proteins was analyzed by western blot with polyclonal anti-s, anti-n, and anti-m antibodies, respectively. the same membrane was also probed with antiactin antibody as a loading control. numbers on the left indicate molecular masses in kilodaltons. rt-pcr amplification of subgenomic mrnas was carried out to check if a low level of subgenomic mrna synthesis could occur in cells transfected with the mutant transcripts. total rna prepared from the transfected cells days postelectroporation was used in the rt reaction with the sense-primer ibv-leader ( ′- ctattacactagccttgcgct - ′) for the detection of negative-stranded sgrna, and the antisenseprimer ibv -r ( ′- , ctctggatccaataacc-tac , - ′) for the detection of positive-stranded sgrna. the two primers were then used for pcr. if transcription of subgenomic mrnas did occur, a bp pcr product corresponding to the ′-terminal region of the subgenomic mrna and a bp fragment corresponding to the ′terminal region of the subgenomic mrna would be expected. as shown in fig. b , a dominant bp band and a weak bp band were observed in cells electroporated with wild type full-length transcripts at days post-electroporation (lanes and ). sequencing of the pcr fragments confirmed that they represent the correct sequences of the corresponding regions of the subgenomic mrnas and , respectively. however, the same pcr products were not detected in cells electroporated with the mutant transcripts (fig. b, lanes and ) . as a negative control, the amplified fragments were not detected in cells without electroporation (fig. b, lane ) . the failure to detect both negative-and positive-stranded sgrnas in cells transfected with the mutant transcripts show that the g c mutation leads to the disruption of subgenomic rna transcription. to further demonstrate that the failure to rescue infectious virus from the g c mutant transcripts is due to a defect in subgenomic rna transcription, the full-length clones with and without the g c mutation were used to generate recombinant ibv expressing the enhanced green fluorescent protein (egfp) by replacing the a gene with egfp. full-length transcripts containing egfp were synthesized in vitro and introduced into vero cells together with the n transcripts by electroporation. at days post-electroporation, single cpe with the expression of egfp was observed in cells transfected with the full-length transcripts without the g c mutation vero cells electroporated with in vitro-synthesized transcripts derived from the in vitro assembled full-length clones containing egfp either with or without g c mutation. phase-contrast and fluorescent images were taken , , and days post-electroporation, respectively. (fig. c) . gradually increased cpe and fluorescent cells were observed from to days post-electroporation (fig. c) . at days post-electroporation, cpe and fluorescent cells were extended to almost the whole monolayer (fig. c) . however, it was consistently observed that much less infectious virus was recovered from cells transfected with this construct. furthermore, the recombinant virus rapidly lost infectivity when passaged on vero cells; the recovered virus maintains minor infectivity only for one passage. in cells transfected with the fulllength transcripts containing the g c mutation, neither cpe nor cells expressing egfp were observed (fig. c) , demonstrating that g c mutation led to total demolition of the egfp expression. as egfp could be expressed only if subgenomic rnas were synthesized but can be observed even if a single cell was transfected and expressed the protein to a certain level, these results reinforce the conclusion that the g c mutation blocks subgenomic rna transcription. mutational analysis of the r residue of the helicase protein g c mutation resulted in the substitution mutation of the r with a pro (r p) of the helicase protein. sequence comparison of the ibv helicase protein with other known coronaviruses showed that r residue is located adjacent to a conserved motif (fig. a) . in all sequenced coronaviruses, only ibv has a charged amino acid (r ) at this position (fig. a) . to assess if a positive charge amino acid at this position is essential for the function of the protein, mutation of r to a lys (r k) was carried out. meanwhile, a conserved leu residue (ile in the case of tgev) was found at this position in all other coronaviruses, mutation of r to a leu (r l) was also included. in vitro full-length transcripts containing the r k and r l mutations were electroporated into vero cells. as shown in fig. b , transcripts generated from wild type (r ) and r k mutant constructs showed very similar infectivity after introduction into vero cells. typical cpe was observed in large areas of the monolayers at days post-electroporation (fig. b) , and recombinant viruses were recovered. however, r l transcripts were found to be less infectious. in cells electroporated with transcripts generated from this mutant, typical cpe was observed in much restricted areas of the monolayer at days post-electroporation (fig. b) . the growth properties of the r k and r l mutant viruses on vero cells were tested by analysis of plaque sizes and growth curves of passage mutant viruses. compared to cells infected with wild type recombinant virus (average plaques size is . ± . ), plaques with similar size were observed in cells infected with the r k mutant virus (fig. a) . the average plaque size in cells infected with r k mutant virus is . ± . mm. in cells infected with the r l mutant virus, much smaller-sized plaques were observed (fig. a) . the average plaque size in cells infected with this mutant virus is . ± . mm. however, analysis of the growth curves of wild type and mutant viruses demonstrated that the mutant viruses exhibited very similar, or even better, growth properties as the wild type recombinant virus (fig. a) . the r k and r l mutant viruses were subsequently characterized by analysis of viral rnas and structural proteins. northern blot analysis showed the detection of very similar amounts of genomic and subgenomic rnas in cells infected with ribv and the two mutant viruses at h post-infection (fig. b) . similarly, western blot analysis of cells infected with ribv, r k, and r l mutant viruses showed that similar amounts of s, n, and m proteins were detected at h post-infection (fig. c, lanes - ) . when probing with anti-n protein antibodies, other species migrating faster than the full-length products on sds-page were also observed (fig. c) . they may represent premature termination and cleavage products of n protein (unpublished observation). it was also noted that variable amounts of these species were detected in cells infected with wild type and different mutants (fig. c) . the genetic stability of r k and r l mutant viruses was tested by propagation of the viruses on vero cells for passages. sequencing analysis of the fifth passages of the two mutant viruses showed that the mutations are stable. no reversion to the original sequences or mutation to other nucleotides was found in the position. in vitro assembly of full-length coronavirus clones, generation of full-length transcripts in vitro using a bacteriophage dna-dependent rna polymerase, and recovery of infectious viruses by introduction of the in vitro-synthesized transcripts into cells, first used by yount et al. ( ) , are a rapid and reliable approach to construct infectious clones from large rna viruses. it has been successfully used to construct infectious fig. . analysis of the growth properties of ribv, r k, and r l mutant viruses. (a) plague sizes and one-step growth curves of ribv, r k, and r mutant viruses. monolayers of vero cells on a -well plate were infected with μl of -fold diluted virus stock and cultured in the presence of . % carboxymethy cellulose at °c for days. the cells were fixed and stained with . % toluidine. to determine the one-step growth curves of ribv, r k, and r l mutant viruses, vero cells were infected with the viruses and harvested at , , , , , , and h postinoculation, respectively. viral stocks were prepared by freezing/thawing of the cells three times, and tcid of each viral stock was determined by infecting five wells of vero cells on -well plates in triplicate with -fold serial dilution of each viral stock. error bar shows standard error of the mean. (b) northern blot analysis of the genomic and subgenomic rnas in cells infected with ribv, r k, and r l mutant viruses. ten micrograms of total rna extracted from vero cells infected with ribv, r k, and r l mutant viruses, respectively, was separated on % agarose gel and transferred to a hybond n+ membrane. viral rnas were probed with a dig-labeled dna probe corresponding to the -end nucleotides of the ibv genome. numbers on the left indicate nucleotides in kilobase, and numbers on the right indicate the genomic and subgenomic rna species of ibv. (c) western blot analysis of viral protein expression in cells infected with wild type and r mutant viruses. vero cells infected with wild type recombinant ibv (lane ), r k (lane ), and r l (lane ) were harvested, lysates prepared, and separated on sds- % polyacrylamide gel. the expression of s, n, and m proteins was analyzed by western blot with polyclonal anti-s, anti-n, and anti-m antibodies, respectively. the same membrane was also probed with anti-actin antibody as a loading control. numbers on the left indicate molecular masses in kilodaltons. clones for several coronaviruses, including transmissible gastroenteritis virus (tgev), mouse hepatitis virus (mhv), sars-cov, and ibv (youn et al., a (youn et al., , b yount et al., yount et al., , . in the process of developing an infectious ibv clone from a vero cell-adapted ibv beaudette strain using this approach, a g-c point mutation at nucleotide position , was found to be lethal to the infectivity of the in vitrosynthesized full-length transcripts on vero cells. no infectious virus could be rescued from vero cells electroporated with transcripts containing this mutation. this mutation causes arg -pro substitution in a domain within the helicase protein (nsp ) with undefined functions, indicating that this domain may be essential for the functionality of the helicase protein. multiple enzymatic activities have been assigned to the nsp helicase protein. these include rna and dna duplex-unwinding activities, ntpase and dntpase activities, and an rna ′-triphosphatase activity that might be involved in the formation of the ′-cap structure of viral rnas (ivanov et al., ) . the protein is comprised of two domains: a putative n-terminal zinc binding domain, which spans the nterminal region of the protein from approximately amino acids to , and a c-terminal helicase domain covering the c-terminal part of the protein from amino acids to the cterminal end (ivanov et al., ) . a ser-pro substitution located immediately downstream of the putative zinc binding domain of nsp , the equivalent rna helicase protein in equine arteritis virus (eav), caused defect in subgenomic mrna transcription (van dinten et al., (van dinten et al., , . more detailed analysis of the zinc binding domain of nsp from eav and nsp from human coronavirus e by mutagenesis studies showed that this domain could modulate the enzymatic activities of the helicase domain (seybert et al., ) . through this regulatory role and some yet to be discovered mechanisms, the zinc binding domain is shown to be critically involved in the replication and transcription of coronavirus rna. in this study, introduction of the in vitro-synthesized fulllength transcripts containing the g c (r p) mutation was shown to be totally defective in subgenomic rna transcription, a phenotype similar to, but appears to be much more severe than, the ser-pro mutation in eav (van dinten et al., (van dinten et al., , . mutation of the arg residue to a positively charged amino acid (lys) does not affect the infectivity of the in vitro-synthesized transcripts as well as the growth properties of the rescued virus. however, mutation of the arg residue to leu, a conserved residue at the same position in most of other known coronaviruses, impaired the recovery rate of the in vitrosynthesized transcripts. the recovered mutant virus showed much smaller-sized plaques. the r residue is located amino acids downstream of the last his (h ) residue in the putative zinc binding domain (fig. a) and amino acids upstream of the helicase motif (seybert et al., ) . so far, no functional domain has been found in this region of the helicase protein. why the r p substitution, located outside of the two functional domains of the helicase protein, shows severe phenotypic defect in subgenomic rna transcription and the infectivity of ibv is not clear at the moment. three possibilities were considered. first, r may be part of the n-terminal zinc binding domain. as no studies have been attempted to define the boundary of the two domains, it is not even certain that an independent domain with unique function may exist in this region. r p mutant virus shares certain phenotypic similarity to the ser-pro mutant eav, such as the absence of subgenomic mrna transcription (van dinten et al., (van dinten et al., , , suggesting that the two mutations might disrupt a similar function of the helicase protein. however, as biochemical characterization of the effect of r p mutation on the enzymatic activities of ibv nsp helicase protein is currently lacking, it would be difficult to draw a conclusion that the two mutants share mechanistically similar characteristics. second, r is located in a region with a high degree of amino acid conservation in all known coronaviruses. since the main defect of r p mutant virus is in the subgenomic rna transcription, one possibility is that this region may be involved in the subgenomic rna synthesis by interacting with host or other viral functional proteins. alternatively, as a positively charged amino acid is required to maintain the full function of the protein, it would be possible that this region may be involved in binding of the helicase protein to viral rna during rna replication and transcription. finally, mutation to a pro would lead to the disruption of the three dimensional structures of the two domains. biochemical characterization of the involvement of this region in the enzymatic activities of the protein and determination of its three dimensional structures are underway to address these possibilities. the g c (e-q) point mutation near the catalytic center of the plp domain in nsp affects neither the recovery of infectious virus from the full-length synthesized in vitro transcripts nor the infectivity of the rescued virus. similarly, efficient recovery of infectious virus from the in vitro transcripts containing the g a (g-e) point mutation in the clp was obtained. the two mutant viruses are genetically stable and show similar growth properties with the wild type recombinant virus. even though the two mutations are located in or near the catalytic centers of the two viral proteinases, the mutations did not alter their enzymatic activities. this relatively high degree of tolerance to mutations in non-essential regions of important functional proteins would minimize the occurrence of lethal mutations during the replication cycles of rna viruses and increase the adaptability of these viruses to a changed environment. vero cells were cultured at °c in minimal essential medium (mem) supplemented with % fetal bovine serum (fbs), penicillin ( units/ml), and streptomycin ( μg/ml). a vero cell-adapted ibv beaudette strain ( passages on vero cells (p )) (shen et al., (shen et al., , fang et al., ) was propagated in vero cells in fbs-free mem. five fragments spanning the entire ibv genome were obtained by rt-pcr from vero cells infected with the vero cell-adapted ibv p at a multiplicity of approximately . briefly, total cellular rna was extracted from the infected vero cells with tri reagent (molecular research center, inc.), according to the manufacturer's instructions. reverse transcription was performed with expand reverse transcriptase (roche) using reverse primers ibv- r, ibv- r, ibv- r, ibv- r, and ibv- r (table ) . each cdna fragment was amplified from rt products by pcr using kod hot start dna polymerase according to the manufacturer's instructions (novagen). the pcr products were purified from agarose gels and cloned into pcr-xl-topo (invitrogen) or pgem-t easy (promage) vectors. subsequently, fragment a was removed from pcr-xl-topo by digestion with nhei and ecori and subcloned into pkt vector. two to three independent clones of each fragment were selected and sequenced by automated sequencing using specific primers and the abi dye termination sequencing method. sequence comparison, assembly, and analysis were performed by using blast and dna star software. mutations were introduced into the corresponding fragments by using quickchange site-directed mutagenesis kit (stratagene) and confirmed by sequencing of the whole fragments. in vitro assembly of full-length cdna clones plasmids were digested with either bsmbi (fragment a) or bsai (fragments b, c, d, and e). the digested plasmids were separated on . % agarose gels containing crystal violet. bands corresponding to each of the fragments were cut from the gels and purified with qiaquick gel extraction kit (qiagen inc.). fragments a and b, and fragments c, d, and e were first ligated with t dna ligase at °c overnight. the two reaction mixtures were then mixed and further ligated at °c overnight. the final ligation products were extracted with phenol/chloroform/isoamyl alcohol ( : : ), precipitated with ethanol, and detected by electrophoresis on . % agarose gels. full-length transcripts were generated in vitro using the mmessage mmachine t kit (ambion, austin, tx) according to the manufacturer's instructions with certain modifications. briefly, μl of transcription reaction with a : ratio of gtp to cap analog was sequentially incubated at . °c for min, . °c for min, . °c min and . °c for min. the n transcripts were generated by using a linearized pkto-ibvn containing ibv n gene and the ′-utr region as templates. a : ratio of gtp to cap analog was used for the transcription of ibv n gene. the in vitro-synthesized full-length and n transcripts were treated with dnasei and purified with phenol/chloroform. vero cells were grown to % confluence, trypsinized, washed twice with cold pbs, and resuspended in pbs. rna transcripts were added to μl of vero cell suspension in an electroporation cuvette and electroporated with one pulse at v, μf with a bio-rad gene pulser ii electroporator. the transfected vero cells were cultured overnight in % fbs-containing mem in a mm dish or a six-well plate and further cultured in mem without fbs. analysis of the negative strand and subgenomic rnas by rt-pcr, real-time pcr total rna was extracted from vero cells electroporated in in vitro-synthesized transcripts after treatment with dnasei, using tri reagent (molecular research center, inc.) at or h post-electroporation. reverse transcription was performed with expand reverse transcriptase (roche) using equal amount of rna. after optimization, real-time pcr was performed using lightcycler faststart dna master sybr green i kit according to the manufacturer's instructions (roche). vero cells were infected with wild type and mutant viruses at a multiplicity of approximately , and total rna was extracted from the infected cells. ten micrograms of rna was added to a mixture of × mops, % formaldehyde, and formamide and incubated at °c for min before subjected to gel electrophoresis. the segregated rna bands were transferred onto a hybond n+ membrane (amersham biosciences) via capillary action overnight and fixed by uv crosslinking (stratalinker). hybridization of dig-labeled dna probes was carried out at °c in hybridization oven overnight. membranes were washed times for min each with the probe buffer, before proceeding to detection with cdp-star (roche) according to the manufacturer's instructions. vero cells were infected with wild type and mutant viruses at a multiplicity of approximately . total proteins extracted from the infected vero cells were lysed with × sds loading buffer in the presence of mm dtt plus mm of iodoacetamide and subjected to sds-page. proteins were transferred to pvdf membrane (stratagene) and blocked overnight at °c in blocking buffer ( % fat free milk powder in pbst buffer). the membrane was incubated with : diluted primary antibodies in blocking buffer for h at room temperature. after washing three times with pbst, the membrane was incubated with : diluted anti-mouse or anti-rabbit igg antibodies conjugated with horseradish peroxidase (dako) in blocking buffer for h at room temperature. after washing three times with pbst, the polypeptides were detected with a chemiluminescence detection kit (ecl, amersham biosciences) according to the manufacturer's instructions. confluent monolayers of vero cells on six-well plates were infected with μl of -fold diluted virus stock. after h of incubation at °c, cells were washed twice with pbs and cultured in ml of mem containing . % carboxymethy cellulose for days. the cells were fixed and stained with . % toluidine. vero cells were infected with wild type and recombinant ibv and harvested at different times post-infection. viral stocks were prepared by freezing/thawing of the cells three times. the % tissue culture infection dose (tcid ) of each sample was determined by infecting five wells of vero cells on -well plates in duplicate with -fold serial dilution of each viral stock. engineering the largest rna virus genome as an infectious bacterial artificial chromosome completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus reverse genetics system for the avian coronavirus infectious bronchitis virus recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism gene of the avian coronavirus infectious bronchitis virus is not essential for replication recombinant mouse hepatitis virus strain a from cloned, full-length cdna replicates to high titers in vitro and is fully pathogenic in vitro selection of and recombination between minor variants lead to the adaptation of an avian coronavirus to primate cells neither the rna nor the proteins of open reading frames a and b of the coronavirus infectious bronchitis virus are essential for replication multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase repair and mutagenesis of the genome of a deletion mutant of the coronavirus mouse hepatitis virus by targeted rna recombination characterization of a papain-like proteinase domain encoded by orf a of the coronavirus ibv and determination of the cterminal cleavage site of an kda protein characterization of the two overlapping papainlike proteinase domains encoded in gene of the coronavirus infectious bronchitis virus and determination of the c-terminal cleavage site of an kda protein identification of a novel cleavage activity of the first papain-like proteinase domain encoded by open reading frame a of the coronavirus avian infectious bronchitis virus and characterization of the cleavage products identification, expression and processing of an k polypeptide encoded by orf a of the coronavirus infectious bronchitis virus proteolytic processing of the coronavirus infectious bronchitis virus a polyprotein: identification of a kda polypeptide and determination of its cleavage sites proteolytic mapping of the coronavirus infectious bronchitis virus b polyprotein: evidence for the presence of four cleavage sites of the c-like proteinase and identification of two novel cleavage products the genome sequence of the sars-associated coronavirus coronavirus reverse genetics by targeted rna recombination identification of a kda polypeptide processed from the coronavirus infectious bronchitis virus a polyprotein by the c-like proteinase and determination of its cleavage sites further characterization of the coronavirus infectious bronchitis virus c-like proteinase and determination of a new cleavage membrane association and dimerization of a cysteinerich, -kda polypeptide released from the c-terminal region of the coronavirus infectious bronchitis virus a polyprotein characterization of a novel coronavirus associated with severe acute respiratory syndrome targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence a complex zinc finger controls the enzymatic activities of nidovirus helicases emergence of an avian coronavirus infectious bronchitis virus (ibv) mutant with a truncated b gene: functional characterization of the b gene in pathogenesis and replication a single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature infectious rna transcribed in vitro from a cdna copy of the human coronavirus genome cloned in vaccinia virus an infectious arterivirus cdna clone: identification of a replicase point mutation that abolishes discontinuous mrna transcription the predicted metal-binding region of the arterivirus helicase protein is involved in subgenomic mrna synthesis, genome replication, and virion biogenesis further identification and characterization of novel intermediate and mature cleavage products released from the orf b region of the avian coronavirus infectious bronchitis virus a/ b polyprotein contribution of trafficking signals in the cytoplasmic tail of the infectious bronchitis virus spike protein to virus infection in vitro assembled, recombinant infectious bronchitis viruses demonstrate that the a open reading frame is not essential for replication 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 reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice this work was supported by the agency for science technology and research, singapore, and by a grant from the biomedical research council (bmrc / / / / ), agency for science, technology and research, singapore. key: cord- -r e bop authors: kassaa, imad al; hober, didier; hamze, monzer; caloone, delphine; dewilde, anny; chihib, nour-eddine; drider, djamel title: vaginal lactobacillusgasseri cmul can inhibit herpes simplex type but not coxsackievirus b e date: - - journal: arch microbiol doi: . /s - - - sha: doc_id: cord_uid: r e bop this study aimed at demonstrating the antiviral activity of lactobacillus gasseri cmul (l. gasseri cmul ), l. acidophilus cmul and l. plantarum cmul against herpes simplex type (hsv- ) and coxsackievirus b e (cvb e ), which are enveloped and naked viruses, respectively. these lactobacilli were non-cytotoxic and were able to reduce the cytopathic effect induced by hsv- in vero cell monolayers. however, lactobacilli were not active against cvb e . tested lactobacilli displayed anti-hsv- activity when they were co-incubated with the virus prior to inoculating the mixture to vero cell monolayers. the detection of hsv- dna by pcr in pellets of bacteria/virus mixtures let us to hypothesize that anti-hsv- activity of lactobacilli resulted from the viruses’ entrapment. this study showed the capabilities of vaginal lactobacilli to inhibit enveloped viruses such as hsv- . activity of probiotic strains. related to this, botić et al. ( ) unveiled the potential activity of probiotic lactobacilli against vesicular stomatitis viruses (vsv). this study shows that antiviral activity is due to the direct interaction between probiotic strains and enveloped virus. attempts to use probiotics as antiviral agents may be a promising alternative (chono et al. ) . probiotic lactic acid bacteria (lab) and bifidobacteria support immune system's function and balance and contribute to immuno-modulatory effects in combating microbial pathogens, including viruses. the herpes infection is recurrent and can be reactivated at any time (field and vere-hodge ) . acyclovir can be potent only in the reactivation stage, and for these reasons the prevention of this type of infection is of major importance in the sector of public health (caldeira et al. ) . taken together, these data indicate that the need of alternative strategies and probiotics could offer various advantages. the vaginal microflora usually designed as vaginal lactobacilli is the first line of defence against a large variety of microbes (al kassaa et al. a, b) . the inhibitory potency of vaginal lactobacilli can be exerted through different mechanisms including the aggregation and production of anti-microbial compounds (al kassaa et al. b ). related to this, lactic acid, hydrogen peroxide and bacteriocins constitute the main anti-pathogen arsenal produced by vaginal lactobacilli (al kassaa et al. a) . conti et al. reported that the anti-hsv- activity of l. brevis was originated by a molecule other than lactic acid and h o (conti et al. ). mastromarino et al. ( ) showed that another strain of l. brevis exerted anti-hsv- activity by a heat-resistant and non-protein cell surface component. subsequent to these findings (mastromarino et al. ) , khani et al. ( ) showed that l. rhamnosus enhanced macrophage viability in elimination of hsv- , while zabihoullahi et al. ( ) reported that supernatants from different lactobacilli inhibited hsv replication. this study aimed at highlighting the anti-hsv- potential of vaginal lactobacilli recently isolated from a cohort of lebanese women. this report focuses, especially on l. gasseri cmul , which is resulting hereafter as able to inactivate the enveloped hsv- but not the naked enterovirus cvb e . of course, the vaginal sampling and research outlines were authorized by the ethical committee of lebanese university as well as lebanese public health authorities. it should be pointed out that all the experiments developed in this study were performed in triplicate and in the independent occasions. the strains used in this study were l. gasseri cmul , l. acidophilus cmul and l. plantarum cmul (al kassaa et al. a ). these strains were grown in mrs broth (de man et al. ) and incubated for h at °c, under anaerobic conditions using anaeropack (biomérieux, france). the cells were harvested ( g, min, °c), and the pellets were washed twice with phosphate buffer solution (pbs) (ph . ). the number of cells was adjusted to cfu/ml by using the turbidity ( nm) and counting method on mrs (de man et al. ) agar plate. staphylococcus aureus (s. aureus) strain was grown in brain heart infusion (biokar, france) at °c for h. the number of colonies was determined on baird-parker agar (biokar, france). the vero cells (kidney epithelial from african green monkey) (atcc ccl- ) were grown in dulbecco's modified eagle's medium (dmem) (gibco, usa), supplemented with % foetal calf serum (gibco, usa), and % l-glutamine (gibco, usa), and incubated at °c in the presence of % co in tissue culture flasks to a confluency of approximately %. further, . × cells/ml were seeded in -well culture cell microplates and incubated under the same conditions to % confluency. the cell viability test was carried out using a commercialized kit "uptiblue" (uptima, interchim, france). briefly, the vero cells were seeded at . × cells/well on -well microtitre plates and cultured until reaching % of confluency. afterwards, the vero cell monolayers were washed twice with pbs and various lactobacilli strains were added on the vero cell monolayers and incubated at °c with % co for h to insure the bacterial adhesion. after bacterial adhesion, the vero cell monolayers were washed three times with pbs followed by addition of µl of uptiblue reagent. this mixture was incubated for h at °c with % co . absorbance was measured on an eia reader (lab systems; mtx labs, vienna, va, usa) at a test wavelength of nm and a reference wavelength of nm. the percentages of cell reductions (cell viability) were determined using the following formula: control well containing bacteria without vero cells, and o.d(c) is the absorbance measured for control untreated mock-infected cells. the possible cytotoxic effect of lactobacilli was measured after , , and h of bacterial adhesion by using the uptiblue kit. therefore, vero cells at % of confluency were incubated with µl of tenfold serial dilutions ( cfu/ml to cfu/ml) and cultured up to h before measuring cell viability. in addition, the presence of bacteria was measured as previously described (jacobsen et al. ) . briefly, a cell suspension containing cells in -ml complete dmem medium was transferred to each well of a six-well tissue culture plate. when the cells reached % confluency, the medium was removed h prior to the adhesion assay and the cells were supplemented with dmem, without antibiotics. the monolayer cells were washed twice with ml of pbs. an aliquot of ml of dmem (without serum and antibiotics) was added to each well and incubated for min at °c. bacterial cells, at cfu/ml, were resuspended in ml of dmem medium (without serum and antibiotics) and added to different wells. the plates were incubated for h at °c in the presence of % co . the monolayer cells were washed five times with sterile pbs, and the adhesion score was determined by counting the adhered bacteria within random microscopic fields after giemsa staining (jacobsen et al. ) . bacteria were grouped into three categories: nonadhesive (≤ bacteria), adhesive ( - bacteria) and highly adhesive (> bacteria). the percentages of adhesion were determined by plating mrs agar plate followed by colony counting after h of incubation. the adhered bacteria were harvested together with the eukaryotic cells by trypsination; the mixture was serially diluted and streaked onto mrs agar plates (jacobsen et al. ). hsv- was isolated at the laboratory of virology (chru lille) from a patient with vaginal infection and was cultured on vero cell line. cvb e is member of the human enterovirus b species of the enterovirus genus that can be grown on hep cell line and on vero cell line as well (sane et al. ) . the supernatants containing the virus were collected from the flask when the cytopathic effect (cpe) was observed by inverted light microscopy (olympus, usa). the viral titres of supernatants were determined by the spaerman-karber tcid ( % tissue culture infectious dose) titration method (landry et al. ) . the plaque reduction assay was carried out as previously described (zabihollahi et al. ) with some modifications. briefly, the vero cells were placed into -well culture plates (nunc, denmark) at a concentration of . × cells per well and incubated for h (in antibiotic free medium) until % of confluency. the vero cells were inoculated with pfu hsv- or tcid /ml cvb e and incubated for h. afterwards, the cell monolayers were washed and were overlaid with dmem, supplemented with . % (w/v) agarose (invitrogen, uk), % fbs, iu/ml penicillin and μg/ml streptomycin (gibco, usa). after h of incubation at °c in % co atmosphere, the overlay medium was removed and the vero cell monolayers washed twice with pbs, followed by fixing with pure methanol for min. counts of the viral plaques was done after staining with . % crystal violet (landry et al. ) . the dna extraction was carried out using the qiaamp minelute virus spin kit (qiagen, germany). the primers and probes targeting hsv- glycoprotein b were designed using the primer express . software (applied biosystems, usa). the primer sequences were fhsv- ′-cgcacctgcgggaaatc- , rhsv- ′-gcgggcacacgtaaa- ′ and taqman probe sequence (hsv- probe): (vic) ′-aggtcgaga acgcc- ′ (mgb). the qpcr was realized on abi (applied biosystems, usa) machine using the following pcr program: min/ °c ( cycle), min at °c ( cycle) and cycles of ( s/ °c, min/ °c). taqman universal master mix ( ×) (applied biosystems, usa) and . μm of primers and probe was used in the qpcrs. dna extracted from hsv- reference strain atcc vr- d was used as control of qpcr. the viability of vero cells decreased to , , and %, after h of incubation in the presence of cfu/ml of l. gasseri cmul , l. acidophilus cmul or l. plantarum cmul and s. aureus atcc , respectively. when the concentration of lactobacilli increased up to cfu/ml after h of incubation, the viability of vero cells was , and % for cmul , cmul and cmul , respectively. however, after h of incubation, the cell viability diminished to and %, in the presence of lactobacilli strains and s. aureus atcc , respectively (fig. ) . taken together, these data indicate that the viability of vero cells was particularly decreasing along time of incubation. the efficiency of adhesion was measured after h of incubation and reached about % for lactobacilli, and % for s. aureus atcc ( table ). the addition of lactobacilli strains to vero cell monolayers followed by their challenge with pfu/ml hsv- reduced the cell death by , and %, respectively, compared with the control that contained vero cells without bacteria and challenged with hsv- (fig. ) . the results of -h lactobacilli contact were quietly different to those obtained after -h contact (fig. ) . there was no significant reduction in vero cells' death when they were challenged with tcid /ml cvb e (fig. ) . no impact on viability of vero cells was observed when l. gasseri cmul was replaced either by l. acidophilus cmul or by l. plantarum cmul (fig. ) . however, when vero cells were infected with hsv- or cvb e and then challenged with lactobacilli at cfu/ml, the observed cytotoxicity remained unchanged (data not shown). the co-incubation of l. gasseri cmul , l. acidophilus cmul or l. plantarum cmul strain with hsv- on vero cell monolayers has increased the cells viability by , and %, respectively, as compared to the control with % viability, after exposure to the virus (fig. a) . in case of l. gasseri cmul , the increase in post-treatment vero cell viability was lactobacilli cell concentration dependent (fig. b) . when the vero cell monolayers were inoculated with cvb e at tcid /ml and lactobacilli at cfu/ml, the cvb e -induced cytopathic effect was not modified. inhibition of the hsv- -induced cytopathic effect by l. gasseri cmul was accompanied by a reduction in viral progeny as compared to the control ( tcid /ml vs. tcid /ml p = . ). however, there was no reduction in viral progeny with strains l. acidophilus cmul ( tcid /ml; p = . ) and l. plantarum cmul ( . tcid /ml; p = . ) (fig. ) . in case of l. gasseri cmul (fig. a) , the dmem supernatant (ph . ), neutralized supernatant (ph . ), and supernatant treated with protease and with catalase to degrade proteins and eliminate possible activity due to the presence of h o caused . , . , . and . log reduction in the number of viral particles, respectively. the dmem lactobacilli supernatants were mixed and incubated with viruses prior to infection step. further, l. gasseri cmul -, l. acidophilus cmul -and l. plantarum cmul -derived neutralized supernatants enhanced slightly the viability of vero cell monolayer inoculated with pfu hsv- with about . , . and . %, respectively, due to reduction in viral infectious particles. these data are normalized to hsv- alone used as a control (table ). this very weak inhibition prompted us to investigate whether the anti-hsv- activity of lactobacilli was exerted through an interaction between bacteria and hsv- particles. thus, l. gasseri cmul at cfu/ml or dulbecco's modified eagle's medium (dmem, negative control) were incubated for h at °c in co atmosphere in the presence of hsv- ( pfu/ml) or cvb e ( tcid /ml). afterwards, supernatant was collected by centrifugation ( , g, min, °c) and assayed on vero cells. after the treatment, the number of infectious hsv- aureus) atcc at cfu/ml. the cell viability was measured using uptiblue, and the results were expressed as the percentage of viability compared to the negative control ( % viable vero cell monolayers). the results were the means (±sd) of at least three independent experiments particles was significantly reduced ( , vs. , pfu/ml p = . ), whereas that of cvb e remained unchanged as compared to the control ( . tcid /ml) (fig. b, c) . to be noted that the pellet was washed three times with dmem in order to eliminate any free residual viral particles in the mixture. after each washing, the supernatants were assayed on vero cells monolayers to evaluate the level of free infectious particles. after the final washing step, dna was extracted from the pellet and the final supernatant to test by qpcr the presence of the viral particles trapped with l. gasseri cmul . thereof, the cycle threshold of the control assay was . ± . for pfu, and that of the pellet was . ± . (fig. d ). in contrast, the detection of hsv- dna in the final washing supernatant was almost negative (fig. d) . the bacterial pellet suspected to be containing hsv- infectious particles, as well as the washing supernatants were serially diluted ( − to − ) and added to vero cell monolayers. afterwards, a pra method was carried out to examine these samples' infectivity, by incubating them for days at °c in % co . after the incubation, no viral infection was observed for the supernatant of the final washing and for the bacterial pellet (fig. e ). vaginal lactobacilli play crucial role in prevention of urogenital infections such as bacterial vaginosis, vaginal candidiasis and viral infections such as those of hsv- (conti et al. ). hsv- is one of the most common sexually hsv- and cvb e were used to challenge vero cells and then incubated for h. the unbound virus particles were removed by three washings. results were recorded after h of incubation and represented by the virus titration and expressed as logtcid /ml, compared to the control cells (medium) which were not treated with lactobacilli. the data (±sd) are the means of at least three independent experiments transmitted infections, acting further as a potential risk factor for acquiring other sexually transmitted infections (chopra et al. ) . here, we demonstrate the anti-hsv- potential of l. gasseri cmul , l. acidophilus cmul and l. plantarum cmul that were recently isolated from human vagina and characterized for their large antagonism and probiotic characteristics (al kassaa et al. a ). the highest anti-hsv- activity was observed for l. gasseri cmul . recently, zabihoullahi et al. ( ) showed the anti-hsv- activity of another vaginal l. gasseri strain. these two independent studies utilized live and non-live l. gasseri, respectively, and revealed the capabilities of these lactobacilli to inhibit hsv- . further, the absence of cytotoxicity of lab probiotics is a prerequisite before undertaking any antiviral study. l. gasseri cmul , l. acidophilus cmul and l. plantarum cmul used in this study are shown to be not cytotoxic for vero cells. safety of bifidobacterium adolescentis spm was established on vero cells , and that of e. faecium on porcine macrophage cell line strain and hsv- mixture was determined using the pra method. e the real-time pcr method was used to detect the presence of "trapped" viruses in the mixture pellet and residual viral particles in washing supernatants. csc crude supernatant of co-incubation, fws first washing supernatant, sws second washing supernatant, tws third washing supernatant. the control wells refer to the wells containing hsv- only, whereas the test wells refer to the wells containing hsv- co-incubated with cmul strain two main pathways (al kassaa et al. b ). the first one is linked to the production of inhibitory metabolites such as lactic acid, bacteriocins and hydrogen peroxide that are endowed with antiviral activity (conti et al. ). here, this possibility is discarded because the assays conducted with the supernatants gathered from l. gasseri cmul , l. acidophilus cmul and l. plantarum cmul cultures revealed either slight or absence of anti-hsv- activity. however, the anti-hsv- activity attributed to molecules of a other than h o and lactic acid bacteria was reported for l. brevis cd , l. salivarius fv and l. plantarum fv isolated from human vagina (conti et al. ). the impact of lactic acid produced by vaginal lactobacilli was shown to interfere with the negative charges of the viral envelopes, enhancing their sensitivity and provoking ease virus inactivation (tao et al. ) . the second possibility of virus inactivation by lab probiotics is thought to be exerted through a physical contact between bacterial cells and virus envelope (al kassaa et al. b) . the data obtained here agree with this concept, as we show that l. gasseri cmul strain is active against enveloped virus (hsv- ), but not against naked one (cbv e ). interestingly, co-incubation of hsv- and l. gasseri cmul experiments as well as the qpcr one let us to think that hsv- particles were indeed present in the bacterial pellet, and the absence of their infectivity could result from trapping/binding mechanism. in direct line, tao et al. ( ) showed the capabilities of lactobacilli to trap human immunodeficiency virus (hiv) by binding the mannose sugar-rich "dome" of their attachment glycoprotein gp . ivec et al. ( ) reported the trapping and adsorption of vesicular stomatitis virus (vsv) by lactobacillus and bifidobacterium species. lai et al. ( ) indicated that the trapping of hiv by vaginal lactobacilli rendered the diffusion of hiv very slower than in the normal conditions. recently, chai et al. ( ) showed the attachment of the enteropathogenic coronavirus to the surface of e. faecium, which might be a means to trap viruses and prevent infection. wang et al. showed that e. faecium inhibited influenza viruses by mechanisms including a direct physical interaction and strengthening of innate defence at the cellular level . the use of lab probiotics as antiviral agents was reported as promising approach for aquaculture (lakshmi et al. ) and poultry industry (seo et al. ) because viral diseases cause an enormous loss in the production in shrimp farms (lai et al. ), and avian influenza virus occurrence is responsible of severe losses to the poultry industry (seo et al. ) . overall, this study shows that hsv- envelope is a key element in the inactivation by vaginal l. gasseri cmul . after the in vivo studies have been established, l. gasseri cmul could be recommended as vaginal probiotic to preventing hsv- infection in healthy women, or to decreasing the period of treatment in the case of infected women. identification of vaginal lactobacilli with potential probiotic properties isolated from women in north lebanon antiviral potential of lactic acid bacteria and their bacteriocins antiviral activity of bifidobacterium adolescentis spm against herpes simplex virus type herpes simplex virus resistance to acyclovir and penciclovir after two decades of antiviral therapy a novel eukaryotic cell culture model to study antiviral activity of potential probiotic bacteria prevalence of herpes simplex virus type and risk factors associated with this infection in women in southern brazil antiviral effects of a probiotic enterococcus faecium strain against transmissible gastroenteritis coronavirus characterization of virus strains resistant to the herpes virus helicaseprimase inhibitor asp (amenamevir) herpes simplex virus : a boon to develop other sexually transmitted infections inhibition of herpes simplex virus type by vaginal lactobacilli a medium for the cultivation of lactobacilli antiviral activity of trappin- and elafin in vitro and in vivo against genital herpes a recent developments in anti-herpesvirus drugs interactions of macrophages with probiotic bacteria lead to increased antiviral response against vesicular stomatitis virus screening of probiotic activities of forty-seven strains of lactobacillus spp. by in vitro techniques and evaluation of the colonization ability of five selected strains in humans in vitro study of the effect of a probiotic bacterium lactobacillus rhamnosus against herpes simplex virus type human immunodeficiency virus type is trapped by acidic but not by neutralized human cervico vaginal mucus probiotics as antiviral agents in shrimp aquaculture a standardized plaque reduction assay for determination of drug susceptibilities of cytomegalovirus clinical isolates antiviral activity of lactobacillus brevis towards herpes simplex virus type : role of cell wall associated components coxsackievirus b can infect human pancreas ductal cells and persist in ductal-like cell cultures which results in inhibition of pdx expression and disturbed formation of islet-like cell aggregates evaluation of leuconostoc mesenteroides yml as a probiotic against low-pathogenic avian influenza (h n ) virus in chickens analysis of lactobacillus products for phages and bacteriocins that inhibit vaginal lactobacilli characterisation of an antiviral pediocin-like bacteriocin produced by enterococcus faecium inhibitory influence of enterococcus faecium on the propagation of swine influenza a virus in vitro inhibition of hiv and hsv infection by vaginal lactobacilli in vitro and in vivo acknowledgments i.a.k. was a recipient of ph.d. fellowship from azm center of biotechnology (lebanon) and lille university (france). the authors declare that there is no conflict of interest. key: cord- - wsyl vk authors: li, wenmiao; xu, cuijing; hao, cui; zhang, yang; wang, zhaoqi; wang, shuyao; wang, wei title: inhibition of herpes simplex virus by myricetin through targeting viral gd protein and cellular egfr/pi k/akt pathway date: - - journal: antiviral res doi: . /j.antiviral. . sha: doc_id: cord_uid: wsyl vk myricetin, a common dietary flavonoid, was reported to possess many different biological activities such as anti-oxidant, anti-inflammatory, and antiviral effects. in this study, we explored the anti-hsv effects and mechanisms of myricetin both in vitro and in vivo. the results showed that myricetin possessed anti-hsv- and hsv- activities with very low toxicity, superior to the effects of acyclovir. myricetin may block hsv infection through direct interaction with virus gd protein to interfere with virus adsorption and membrane fusion, which was different from the nucleoside analogues such as acyclovir. myricetin also down-regulate the cellular egfr/pi k/akt signaling pathway to further inhibit hsv infection and its subsequent replication. most importantly, intraperitoneal therapy of myricetin markedly improved mice survival and reduced virus titers in both lungs and spinal cord. therefore, the natural dietary flavonoid myricetin has potential to be developed into a novel anti-hsv agent targeting both virus gd protein and cellular egfr/pi k/akt pathway. herpes simplex virus type (hsv- ) and type (hsv- ) are enveloped double stranded dna viruses belonging to herpesviridae (fatahzadeh and schwartz, ) . hsv infection causes skin lesions that are generally localized at the oral, nasal, and ocular level with hsv- infection, whereas with hsv- , infections most commonly occur at genital-skin and mucosa sites (liu and cohen, ; smith and robinson, ) . approximately %- % of the adult human population worldwide is sera-positive for hsv- (carr et al., ) . the current fda approved antiviral agents for herpesviruses involve mainly nucleoside analogues, such as acyclovir (acv) and pencyclovir, which mainly inhibit viral genome replication. despite these successes, drug resistance, and side effects remain unresolved issues in the fight against hsv infections (morfin and thouvenot, ) . hence, the development of novel anti-hsv agents with active mechanisms different from nucleoside analogues is of high importance. myricetin ( , , -trihydroxy- -( , , -trihydroxyphenyl)- -benzopyrone) is a common dietary flavonoid from plant sources such as vegetables, fruits, and tea, with antioxidant properties (ong and khoo, ; ross and kasum, ) . like other flavonoids, myricetin is reported to possess many different biological activities such as antimicrobial, anti-thrombotic, neuroprotective, and anti-inflammatory effects (cushnie and lamb, ; dajas et al., ; gupta et al., ; ong and khoo, ; santhakumar et al., ; semwal et al., ; tian et al., ) . pasetto et al. reported that myricetin possessed anti-hiv- activities with low toxicity, and it mainly inhibited the activity of hiv reverse transcriptase (pasetto et al., ) . lyu et al. found that some flavonoids such as myricetin and quercetinin showed moderate inhibitory effects against hsv- in vero cells (lyu et al., ) . yu and co-workers found that myricetin and scutellarein potently inhibited the sars-cov helicase protein in vitro by affecting the atpase activity, suggesting that myricetin and scultellarein might serve as sars-cov chemical inhibitors (yu et al., ) . thus, myricetin has the potential to be developed into a novel anti-viral agent. to further correlate the anti-viral applications of myricetin with its underlying molecular mechanisms, the anti-hsv effects and mechanisms of myricetin were investigated both in vitro and in vivo in this study. the results showed that myricetin possessed anti-hsv- and hsv- activities with very low toxicity. myricetin may block hsv https://doi.org/ . /j.antiviral. . received october ; received in revised form december ; accepted january infection through direct interaction with virus gd protein to interfere with virus adsorption and membrane fusion. moreover, intraperitoneal therapy of myricetin markedly improved mice survival and reduced virus titers in both lungs and spinal cord. thus, myricetin merits further investigation as a novel anti-hsv agent in the future. myricetin (with purity > %) was purchased from topscience co., ltd. (shanghai, china) . acyclovir was purchased from sigma aldrich (st. louis, mo, usa). vero, hela and hep- cells were routinely cultured in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (excell bio, china), penicillin ( u/ml), and streptomycin ( μg/ml) at °c in % co . hsv- strain f was purchased from atcc (vr- ). hsv- strain was obtained from wuhan institute of virology, chinese academy of sciences. the antiviral activity was evaluated by the cpe inhibition assay (dai et al., ) . briefly, vero cells in -well plates were infected with hsv- or hsv- at a multiplicity of infection (moi) of . , and then treated with indicated concentrations of myricetin in triplicate after removal of virus inoculum. after h incubation, the cells were fixed with % formaldehyde for min at room temperature (rt). after removal of the formaldehyde, the cells were stained with . % (w/v) crystal violet for min at °c. the plates were washed and dried, and the intensity of crystal violet staining for each well was measured at nm. the concentration required for a test compound to reduce the cpe of hsv by % (ic ) was determined. hsv- or hsv- ( - pfu/well) was pre-incubated with or without myricetin for min at °c before infection. then the virusmyricetin mixture was transferred to vero cell monolayers in -well plates, and incubated at °c for h with gentle shaking every min. after that, the inoculum was removed and each well was overlaid with ml of agar overlay media containing . % agarose, u/ml penicillin, and μg/ml streptomycin. the cells were then incubated at °c until plaque sizes were adequate. then the cells were fixed with % paraformaldehyde (pfa) and stained with % crystal violet for plaque counting. vero cells were infected with hsv- or hsv- (moi = . ) under four different treatment conditions: i) pretreatment of virus: myricetin ( μm) pretreated hsv was added to vero cells and incubated at °c for h. then after adsorption, the virus innuculum containing myricetin was removed and the cells were overlaid with compound-free media. ii) pretreatment of cells: vero cells were pretreated with μm of myricetin before hsv infection. iii) adsorption: vero cells were infected in media containing myricetin ( μm) at °c for h. after that, the virus innuculum was removed and the compound-free media were added into cells. iv) post-adsorption: after removal of unabsorbed virus, myricetin ( μm) was added to the cells. at h p.i., virus yields were determined by plaque assay. the indirect immunofluorescence assay was performed as previously described . briefly, for hsv binding assay, hsv- (moi = . ) was adsorbed to hela cells for h at °c in the absence or presence of μm myricetin before washing with pbs. for entry assay, hsv- (moi = . ) was firstly adsorbed to hela cells at °c for h before washing with pbs. then myricetin ( μm) was added to cells and incubated at °c for h. after that, cells were treated with proteinase k to remove adsorbed but not internalized virus. then the cells were washed with pbs and fixed with % paraformaldehyde for min. then cells were permeabilized using . % (v/ v) triton x- in pbs for min before incubated with % bsa for h at °c. after washing, cells were incubated consecutively with anti-icp antibodies ( : dilutions) and dylight ®-conjugated secondary antibodies. then the cell nucleus was stained with dapi for min before confocal imaging. images were recorded using a nikon confocal microscope, and analysed by imagej (nih) version . u (usa). the total rna was extracted from hsv infected vero cells using an rnaiso™ plus kit (takara, japan), and analysed by using the one step sybr primescript rt-pcr kit (takara, japan). the primer pairs for hsv gd and cellular β-actin mrna were listed as follows: gd mrna, ′-agcatcccgatcactgtgtacta- ′ and ′-gcgatggtcaggttgt acgt- '; monkey β-actin mrna, ′-ctccatcctggcctcgctgt- ′ and ′-gctgtcaccttcaccgttcc- '. the real-time rt-pcr was performed at °c min, °c s, cycles of °c s, °c s, followed by melting curve analysis, according to the instrument documentation (abi prism , applied biosystems, usa). all reactions were performed in triplicate and the results were normalized to β-actin. the relative amounts of hsv gd mrna molecules were determined using the comparative ( -ΔΔct ) method, as previously described (livak and schmittgen, ) . darts experiments for identifying the targets of myricetin were performed as previously reported (lomenick et al., ). briefly, hsv- (moi = . ) infected vero cells were lysed with np- cell lysis buffer (beyotime, nantong, china) and treated with or without myricetin ( μm) for h at room temperature (rt) followed by digested with pronase ( μg/ml) in reaction buffer ( mm tris-hcl (ph . ), mm nacl, mm cacl ) for min at rt. the digestion was stopped by directly add × sds-page loading buffer and inactivation by boiling. protein samples were separated with % sds-polyacrylamide gels and analysed by western blot with antibodies against hsv- gd, gb, gc, gh/gl proteins or cellular hvem, nectin- , actin, tubulin proteins as controls. spr assays were conducted on a spr biosensor instrument ge biacoret (ge, usa). hsv- gd proteins (biodragon, beijing, china) were firstly immobilized onto the surface of a carboxymethylated dextran sensor chip (cm ) via amino group coupling as described previously (geng et al., ) . to assess real-time binding of myricetin to the gd proteins on cm chips, myricetin with different concentrations ( , , . , . , . , . μm) dissolved in dmso, was injected over the sensor chip surface with gd immobilized within min, followed by a -min wash with × pbst buffer. the sensor chip surface was then regenerated by washing with naoh ( mm) for s. all binding experiments were carried out at °c with a constant flow rate of μl/s pbs buffer. to correct for non-specific binding and bulk refractive index change, a blank channel without gd was used and run simultaneously for each experiment. then, the biacoret spr evaluation software was used to calculate the kinetic parameters, and the changes in mass due to the binding response were recorded as w. li, et al. antiviral research ( ) resonance units (ru). after drug treatment, the cell lysate was separated by sds-page and transferred to nitrocellulose membrane. after being blocked in trisbuffered saline (tbs) containing . % tween (v/v) and % bsa (w/ v) at rt for h, the membranes were rinsed and incubated at °c overnight with primary antibodies against phosphorylated or nonphosphorylated egfr, pi k, akt antibodies, or anti-α-tubulin and gapdh antibodies (cell signaling technology, danvers, usa) as control. the membranes were washed and incubated with ap-labeled secondary antibody ( : dilutions) at rt for h. the protein bands were then visualized by incubating with the developing solution (pnitro blue tetrazolium chloride (nbt) and -bromo- -chloro- -indolyl phosphate toluidine (bcip)) at rt for min. the relative densities of proteins were all determined by using imagej (nih) v. . u (usa). the cell fusion inhibition assay was performed as described with modifications (du et al., ) . monolayers of vero cells grown in well plates were infected with hsv- (moi = . ) at °c for h. after removal of virus inoculum, compound free media were added to cells and incubated at °c for h. then myricetin ( or μm) was added to cells, and incubated at °c for another h. after that, cells were fixed and detected with hematoxylin-eosin (he) staining. the inhibition of syncytium formation was examined by light microscopy. the influence of myricetin on hsv glycoprotein expression during - h post infection was also evaluated by western blotting. molecular docking was conducted in moe v . (moe, ) . the d structures of myricetin was drawn in chembiodraw and converted to d structure in moe through energy minimization. the d structure of the protein gd was downloaded from rcsb protein data bank (pdb id: myv). prior to docking, the force field of amber : eht and the implicit solvation model of reaction field (r-field) were selected. moe-dock was used for molecular docking simulations of myricetin with gd. the docking workflow followed the "induced fit" protocol, in which the side chains of the receptor pocket were allowed to move according to ligand conformations, with a constraint on their positions. the weight used for tethering side chain atoms to their original positions was . for each ligand, all docked poses of which were ranked by london dg scoring first, then a force field refinement was carried out on the top poses followed by a rescoring of gbvi/wsa dg. the conformations with the lowest free energies of binding were selected as the best (probable) binding modes. molecular graphics were generated by pymol. all animal experiments were performed in accordance with the national institutes of health guide for the care and use of laboratory animals and approved by the institutional animal care and use committee at ocean university of china. three-week-old female balb/ c mice (average weight, . ± . g) were purchased from beijing vital river laboratory animal technology co., ltd. (beijing, china) and raised in a pathogen-free environment ( ± °c and % ± % humidity). mice were randomly divided into five experimental groups ( mice each). mice were anaesthetized and infected via the intranasal route with pfu of hsv- (f strain) in μl of pbs. four hours after inoculation, mice received intraperitoneally treatment of acyclovir ( mg·kg- ), myricetin ( . or mg·kg- ), or placebo, and the treatments were repeated once daily for five days. each day mice were weighed and monitored for signs of illness for days, and those suffering a severe infection or having lost > % of their original body weight were euthanized. to determine virus titers in organs, mice were euthanized, and the lungs, brains and spinal cords were removed, homogenized and clarified by centrifugation on day after inoculation. samples were assayed for virus titers by plaque assay and quantitative rt-pcr assay. histopathological analysis was also performed using h&e staining on lung and brain samples collected on day . all data are representative of at least three independent experiments. data are presented as means ± standard deviations (s.d.). statistical significance was analysed using graphpad prism software using oneway anova with turkey's test. p values < . were considered significant. myricetin is a member of the flavonoid class of polyphenolic compounds, with antioxidant properties, and its structure is shown in fig. a . herein, the anti-hsv effects and mechanisms of myricetin were investigated in vitro. the cytotoxicity of myricetin in vero, hep- and hela cells was firstly assessed by mtt assay (livak and schmittgen, ) . the results showed that myricetin exhibited no significant cytotoxicity at the concentrations from to μm (fig. b) . the cc ( % cytotoxicity concentration) value for myricetin in vero, hep- and hela cells was about . ± . μm, . ± . μm, and . ± . μm, respectively (table ) . myricetin was then assayed for its ability to inhibit hsv multiplication in vitro using cpe inhibition assay and plaque assay . briefly, hsv- (f strain) or hsv- ( strain) (moi = . ) was pretreated with myricetin ( . , . , , μm) for h at °c before infection, then after adsorption, the media containing myricetin at indicated concentrations were added to cells. at h p.i., the viral titers in the culture media were determined by plaque assay, and cell viability was measured by cpe inhibition assay. as shown in fig. c , myricetin significantly reduced the virus titers of both hsv- and hsv- in vero cells when used at the concentration > . μm (p < . ). cpe inhibition assay showed that the ic values of myricetin was about . ± . μm and . ± . μm for hsv- and hsv- , respectively, and the selectivity index (cc /ic ) for myricetin was about . and . , respectively, superior to that of acyclovir (acv) (si = . and . ) ( table ) . myricetin also inhibited hsv replication in hep- and hela cells with si > (table ). in addition, the plaque reduction assay showed that pretreatment of hsv with myricetin at the concentrations of . - μm significantly reduced the number of plaques in both hsv- and hsv- infected cells (fig. d-f) , suggesting that myricetin may be able to inactivate viral particles directly. the time-of-addition assay was performed to determine the stage(s) at which myricetin exerted its inhibition actions in vitro . in brief, myricetin was added to vero cells under four different conditions: pre-treatment of viruses, pre-treatment of cells, during virus adsorption, or after adsorption. subsequently, the antiviral activity was determined by plaque assay. as shown in fig. a , pretreatment of hsv- or hsv- with μm myricetin for h before infection significantly reduced the virus titers of hsv, compared to the non-treated virus control group (p < . ), suggesting that myricetin may have direct w. li, et al. antiviral research ( ) interaction with hsv particles. treatment of myricetin during adsorption or after adsorption also possessed obvious inhibition on virus multiplication (p < . ) ( fig. a) . however, pretreatment of cells did not significantly reduce virus titers in vitro ( fig. a) , suggesting that myricetin may not interact with vero cells directly. moreover, the indirect immunofluorescence assay under these four conditions (pachota et al., ) indicated that pretreatment of virus with myricetin ( μm) before infection also significantly reduced the expression of icp in hsv- infected cells (fig. b) , which was in concern with the results in plaque assay ( fig. a) . thus, myricetin may interact with virus particles to block the adsorption of hsv or inhibit some steps after virus adsorption. since myricetin may be able to block adsorption and post-adsorption processes of hsv, we further explored the potential inhibition of myricetin on hsv binding and entry process by using immunofluorescence assay. briefly, for hsv binding assay, hsv- (moi = . ) was adsorbed to hela cells for h at °c in the absence or presence of μm myricetin before washing with pbs. for entry assay, hsv- (moi = . ) was firstly adsorbed to hela cells at °c for h before washing with pbs. then myricetin ( μm) was added to cells and incubated at °c for h. after that, cells were treated with proteinase k to remove adsorbed but not internalized virus. then the immunofluorescence assay was performed to determine the amount of hsv virions binding to or entering into hela cells. as shown in fig. c and e, myricetin treatment ( μm) during adsorption significantly decreased the fluorescence of icp protein on cell surface, compared to that in non-treated virus control cells, suggesting that myricetin may block virus adsorption process of hsv. moreover, at h p.i., many fluorescence spot of icp could be found at the nucleus area (white arrow indicated), suggesting that hsv- nucleocapsid had been egressed from late endosomes and delivered to the nuclear periphery in hela cells (fig. d) . however, myricetin treatment during entry process dramatically reduced the fluorescence of icp in cytoplasm, and only very little fluorescence could be observed in cell nucleus, suggesting that myricetin may also be able to block hsv entry process in hela cells (fig. d, f and g ). thus, myricetin may possibly block hsv infection mainly through interfering with hsv binding and entry process. since myricetin may block hsv binding and entry process, we then asked whether myricetin could inhibit the virus-induced membrane fusion process. as shown in fig. a , in hsv- (moi = . ) infected vero cells, obvious syncytia with multinuclear cells were observed in non-treated virus control group (hsv- ) at h p.i. however, treatment with myricetin ( , μm) during - h p.i. markedly blocked syncytium formation only with a limited number of small syncytia, suggesting that myricetin may inhibit hsv-induced cell fusion. in addition, myricetin treatment during - h p.i. did not obviously influence the expression of virus surface glycoproteins such as gd protein (fig. b) . thus, myricetin may directly block hsv-induced membrane fusion through inhibiting the function of virus surface glycoproteins rather than reducing their expression. since myricetin can inhibit both hsv adsorption and membrane fusion, we then questioned whether myricetin directly interacts with virus surface gd protein which is mainly required for hsv entry process. we employed a drug affinity responsive target stability assay (darts) (lomenick et al., ) , which relies on the reduction of protease susceptibility of the target protein upon drug binding, to detect the potential interaction between myricetin and gd protein. in brief, hsv- infected vero cell lysates were digested with pronase in the presence or absence of myricetin ( μm), then the amount of gd protein in cell lysates was detected by western blotting. as shown in fig. c , hsv- gd protein was obviously protected by protease digestion in the extracts of myricetin ( μm) treated cells especially under pronase treatment at μg/ml, suggesting that myricetin may interact with hsv gd protein in hsv- infected vero cells. however, myricetin ( μm) could not protect other virus surface glycoproteins such as gb, gc, and gh/gl proteins from protease digestion in hsv- infected cells (fig. d) . similarly, the cellular surface receptors of hsv- such as hvem and nectin- could also not be protected from protease digestion by myricetin ( μm) (fig. d) . thus, myricetin may interfere with hsv binding and entry process through targeting virus gd protein rather than cellular receptors of hsv. moreover, the direct interaction between myricetin and gd protein was further evaluated by using spr assay (geng et al., ) . briefly, with hsv- gd proteins being immobilized on the chip, myricetin at the concentrations of . - μm was flowed over the biosensor chip surface, respectively. data revealed a marked binding of myricetin to virus gd protein in a concentration-dependent manner with a kd equivalent to about . e- m ( . nm), implicating a high affinity of myricetin for hsv- gd protein (fig. e) . thus, pretreatment of hsv were infected with hsv- or hsv- (moi = . ) using four different treatment conditions. i) pre +virus: hsv was pretreated with myricetin ( μm) at °c for h before infection. ii) pre+cell: vero cells were pretreated with μm of myricetin before infection. iii) adsorption: vero cells were infected in media containing myricetin ( μm) at °c for h. iv) post-adsorption: after removal of unabsorbed virus, myricetin ( μm) was added to the cells. at h p.i., virus yields were determined by plaque assay. the results were presented as mean ± s.d. from five independent experiments. *p < . vs. virus control group. (b) vero cells were infected with hsv- (f strain) under four treatment conditions of myricetin, and the expression of virus icp protein was detected by immunofluorescence assay. scale bar represents μm. (c and d) hsv- (moi = . ) infected hela cells were treated with or without myricetin ( μm) during adsorption process (c) or entry process (d), then after washing three times with pbs, the immunofluorescence assay was performed by using anti-icp antibody to evaluate the amount of virions binding to or entering into cells. scale bar represents μm. (e-g) the average fluorescence intensity of icp proteins during adsorption process (e) or entry process (f) was measured by imagej (nih) version . u (usa) to calculate the average intensity per cell of different images (n = ). the average intensity of icp in cell nucleus during entry process (g) was also measured by imagej (usa) to calculate the average intensity per unit area of cell nucleus of different images (n = ). the average intensity for nontreated virus control cells (hsv) was assigned values of and the data presented as mean ± s.d. (n = ). significance: **p < . vs. virus control group (hsv). w. li, et al. antiviral research ( ) with myricetin before infection may allow myricetin to fully bind gd protein and form a stable myricetin-gd complex. furthermore, the impact of myricetin on hsv- gd binding to vero cells was further explored using a cell enzyme-linked immunosorbent assay, as previously described (tiwari et al., ) . briefly, vero cells were first treated with or without myricetin ( , , , μm) for h, then the hsv- gd protein or myricetin ( , , , μm) pretreated gd protein ( μg/ml) were added to cells and incubated for another h, the hsv- gd proteins were firstly immobilized onto the surface of a carboxymethylated dextran sensor chip (cm ). to assess realtime binding of myricetin to the gd proteins on cm chips, myricetin ( , , . , . , . , . μm) was flowed over the biosensor chip surface. the sensorgram for all binding interactions were recorded in real time and were analysed after subtracting the sensorgram from the blank channel. then, the changes in mass due to the binding response were recorded as resonance units (ru). (f) hsv- gd binding assay. vero cells were first treated with or without myricetin ( , , , μm) for h, then the hsv- gd protein or myricetin ( , , , μm) pretreated gd protein ( μg/ml) were added to cells and incubated for another h, respectively. then the amount of gd protein binding to vero cell surface was detected by elisa assay using anti-gd antibody and hrp labeled secondary antibody sequentially. values are means ± sd (n = ). significance: **p < . vs. virus control group. (g and h) the d (g) and d (h) binding mode of myricetin with hsv gd protein (pdb id: myv) was shown. myricetin is colored in purple, the surrounding residues in the binding pockets are colored in cyan. the backbone of the receptor is depicted as lightblue cartoon. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) w. li, et al. antiviral research ( ) respectively. then the amount of gd protein binding to vero cell surface was detected by sequentially incubated with anti-gd antibody and hrp labeled secondary antibody. as shown in fig. f , pre-incubation of gd protein with myricetin ( , , , μm) significantly reduced the binding of gd to cell surface in a dose-dependent manner, however, pretreatment of cells did not significantly decrease the amount of gd protein on cell surface. this finding strongly supports that myricetin may interact with hsv gd protein rather than cellular receptors to block virus binding and entry. to further investigate the binding mode of myricetin with gd, docking simulation studies were carried out using the crystal structure of hsv- gd protein (pdb code: myv). the results indicated that the oxygen atom of carbanyl group of myricetin, regarded as hydrogen bond acceptor, forms two hydrogen bonds with the nitrogen atom of amide group of gln as well as asn in gd (fig. g) . the oxygen atom of phenolic hydroxyl group of myricetin, regarded as hydrogen bond donor, forms a hydrogen bond with the oxygen atom of backbone of leu in gd. the oxygen atom of hydroxyl group of myricetin, regarded as hydrogen bond donor, forms a hydrogen bond with the oxygen atom of carboxyl group of glu in gd ( fig. g and h) . thus, myricetin may interact with gln , leu , glu and asn of gd through hydrogen bond interactions. since myricetin may inhibit hsv entry process in hela cells, we further explored whether myricetin could influence the hsv infectionrelated signaling pathways by using western blot assay. the egfr/ pi k/akt signaling pathway was reported to be required for virus endocytosis and replication, and the inhibitors of pi k signaling could inhibit both entry and fusion of hsv (eierhoff et al., ; tiwari and shukla, ) . herein, treatment with myricetin ( , μm) for h significantly decreased the levels of phosphorylated egfr from about . to about . and . -fold of normal control group, respectively (p < . ) (fig. a and d) . moreover, the activation of downstream signal pi k was also evaluated, and the results showed that treatment with myricetin ( , , μm) for h significantly decreased the levels of phosphorylated pi k proteins from about . to about . , . , and . -fold of normal control group, respectively (p < . ) (fig. b and e). in addition, treatment with myricetin ( , , μm) for h significantly reduced the activation of akt from about . to about . , . , and . -fold of normal control group, respectively (p < . ) ( fig. c and f) . however, myricetin ( . , , , μm) treatment did not significantly influence the total expression levels of egfr, pi k and akt proteins in hsv- infected vero cells (fig. g and h) . thus, cellular egfr/pi k/akt signaling pathway may be involved in the anti-hsv fig. . involvement of egfr/pi k/akt signaling pathway in the anti-hsv actions of myricetin. (a-c) hsv- (moi = . ) infected cells were treated with or without myricetin ( . , , , μm) for h, and then the phosphorylation of egfr (a), pi k (b), and akt (c) was evaluated by western blot. blots were also probed for gapdh and α-tubulin proteins as loading controls. the result shown is a representative of three separate experiments. (d-f) quantification of immunoblot for the ratio of p-egfr (d), p-pi k (e), p-akt (f) protein to gapdh or tubulin, respectively. the ratio for non-infected cells (m) was assigned values of . and the data presented as mean ± s.d. (n = ). significance: ##p < . vs. normal control group (mock); *p < . , **p < . vs. virus control group (hsv). (g) hsv- (moi = . ) infected cells were treated with or without myricetin ( . , , , μm) for h, and then the total expression levels of egfr, pi k, and akt were evaluated by western blot. the result shown is a representative of three separate experiments. (h) quantification of immunoblot for the ratio of egfr, pi k, akt protein to α-tubulin, respectively. the ratio for non-infected cells (mock) was assigned values of . and the data presented as mean ± s.d. (n = ). w. li, et al. antiviral research ( ) actions of myricetin in vitro. furthermore, myricetin ( . , , , μm) treatment could not significantly influence the activation of egfr, pi k and akt proteins in the non-infected vero cells ( fig. a and b) , suggesting that the inhibition of egfr/pi k/akt pathway by myricetin may be related to its inhibition of hsv infection. moreover, treatment with hsv- gd protein ( μg/ml) could significantly enhance the phosphorylation of egfr, pi k, and akt proteins in the non-infected vero cells (p < . ), suggesting that the activation of egfr/pi k/akt pathway in hsv infected cells may be related to the binding of gd to cellular receptors ( fig. c and d) . however, after myricetin treatment ( , , μm), the levels of phosphorylated egfr, pi k, and akt proteins were significantly reduced as compared to the gd treated control group (p < . ) ( fig. c and d) . in addition, myricetin treatment ( , , μm) also significantly reduced the expression level of gd mrna in hsv- and hsv- infected vero cells (p < . ) ( fig. e and f) . considered that egfr/pi k/akt pathway is required for efficient hsv replication, we suppose that myricetin may be able interact with virus gd protein to interfere with the activation of egfr/pi k/akt pathway to further inhibit subsequent replication of hsv. the anti-hsv effects of myricetin were further tested in a murine intranasal model of hsv pneumonia and encephalitis (de clercq and luczak, ) . in brief, hsv- (f strain)-infected mice received intraperitoneal treatment of myricetin ( . or mg·kg- ), acyclovir ( mg·kg- ), or placebo (pbs) once daily for five days. as shown in fig. a , intraperitoneal treatment of myricetin ( . or mg·kg- ) significantly increased survival rates as compared to the placebotreated control group (p < . ). by day post infection, only % of the individuals in the placebo group survived whereas % of animals in the myricetin-treated group survived, superior to that in acyclovir ( mg·kg- )-treated group ( %). moreover, the body weights of mice in virus control group (placebo) began to decrease at days p.i., losing up to % of initial weight, before gradually recovering. however, myricetin ( . or mg·kg- )-treated mice gradually increased their body weights only with a limited weight loss of less than % during the infection, superior to those in the acyclovir ( mg·kg- ) treated group (fig. b) . furthermore, four days post-infection, four mice of each treatment group were sacrificed and the tissue samples were removed for further analysis. subsequently, the viral titers in the lung and spinal cord were determined by plaque assay and quantitative rt-pcr. the results showed that after treatment of myricetin for four days, the pulmonary virus titers significantly decreased compared to that of the virus control group (p < . ) (fig. c ). the relative virus gd mrna levels in spinal cord also significantly reduced compared to that of the placebo group, suggesting that intraperitoneal therapy with myricetin could inhibit hsv multiplication in mice (fig. d) . acyclovir ( mg·kg- ) treatment also showed significant reduction of virus titers in mice lungs and spinal cord ( fig. c and d) . to further evaluate the effects of myricetin on viral pneumonia and encephalitis in mice, histopathology analysis was also performed. the results showed that the lung tissues in virus-control group showed marked infiltration of inflammatory cells and the presence of massive hyperaemia in the lumen (fig. e) . however, mice treated with myricetin ( . or mg·kg- ) following infection had intact columnar epithelium even in the presence of tiny serocellular exudates in the lumen fig. . the inhibition of egfr/pi k/akt pathway by myricetin may be related to its inhibition of gd protein. (a) non-infected vero cells were treated with or without myricetin ( . , , , μm) for h, and then the phosphorylation of egfr, pi k, and akt was evaluated by western blot. blots were also probed for α-tubulin proteins as loading controls. the result shown is a representative of three separate experiments. (b) plots quantifying the immunoblots (as ratios to tubulin) for p-egfr, p-pi k and p-akt proteins. the ratios for non-treated cells (mock) were assigned values of . and the data presented as mean ± s.d. (n = ). (c) vero cells were treated with or without gd proteins in the presene or absence of myricetin ( , , μm) for h, and then the phosphorylation of egfr, pi k, and akt was evaluated by western blot. blots were also probed for gapdh proteins as loading controls. the result shown is a representative of three separate experiments. (d) quantification of immunoblot for the ratio of p-egfr, p-pi k, p-akt protein to gapdh, respectively. the ratio for non-treated control cells (mock) was assigned values of . and the data presented as mean ± s.d. (n = ). significance: ##p < . vs. normal control group (mock); *p < . , **p < . vs. gd treated control group (gd). (e and f) hsv (moi = . ) infected vero cells were treated with myricetin ( . , , , μm), and incubated at °c for h. after that, total rna was extracted for real-time rt-pcr assay of hsv- (e) and hsv- (f) gd mrnas and cellular β-actin mrna. the relative amounts of hsv gd mrnas were determined using the comparative ( -ΔΔct ) method. rna levels for non-treated virus control cells (hsv) were assigned values of . . values are means ± sd (n = ). significance: *p < . , **p < . vs. virus control group. w. li, et al. antiviral research ( ) fig. . the anti-hsv actions of myricetin in vivo. (a) survival rate. hsv- infected mice were received intraperitoneally treatment of myricetin ( . or mg·kg- ), acyclovir ( mg·kg- ), or placebo (pbs) once daily for five days. results are expressed as percentage of survival, evaluated daily for days. significance: *p < . , **p < . vs. virus control group (placebo). (b) hsv infected mice were received intraperitoneally treatment with indicated compounds for five days. the average body weights in each group were monitored daily for days and are expressed as a percentage of the initial value. (c and d) viral titers in lungs and spinal cord. after intraperitoneal therapy with indicated compounds for five days, the viral titers in lungs (c) and spinal cords (d) were evaluated by plaque assay and quantitative rt-pcr assay on day p.i., respectively. values are means ± s.d. (n = ). significance: *p < . , **p < . vs. virus control group. (e and f) histopathology analyses of lung and brain tissues on day p.i. by he staining ( × and × ). the representative micrographs from each group were shown. mock: non-infected mice tissues; hsv- : hsv- infected tissues without drugs; hsv- +acyclovir- : hsv- infected tissues with acyclovir ( mg·kg- ) treatment; hsv- +myricetin- : hsv- infected tissues with myricetin ( mg·kg- ) treatment; hsv- +myricetin- . : hsv- infected tissues with myricetin ( . mg·kg- ) treatment; the black arrows indicate the presence of hyperaemia and infiltration of inflammatory cells in the lumen. ( fig. e ). in addition, treatment of myricetin ( mg·kg- ) nearly had no hyperaemia and infiltration of inflammatory cells in brain tissues (fig. f) . myricetin ( . mg·kg- ) treated mice only had tiny hyperaemia in the brain tissues, comparable to the effects of acyclovir ( mg·kg- ) (fig. f) . thus, myricetin may be able to attenuate pneumonia and encephalitis symptoms in hsv infected mice. myricetin is a member of the flavonoid class of polyphenolic compounds, which was reported to possess antiviral activities against lots of viruses including hiv and sars-cov (ong and khoo, ) . in this study, we discovered that myricetin possessed anti-hsv- and hsv- activities in different cells with very low toxicity (si > ). myricetin may block hsv infection through direct interaction with virus gd protein to interfere with virus adsorption and membrane fusion, which was different from the nucleoside analogues such as acyclovir. most importantly, intraperitoneal therapy of hsv- -infected mice with myricetin markedly improved their survival and reduced virus titers in both lungs and spinal cord. pretreatment of hsv by myricetin before infection significantly reduced the virus titers in hsv infected cells, suggesting that myricetin may have direct interaction with hsv particles. in addition, myricetin can block hsv induced membrane fusion and interfere with virus binding and entry process, suggesting that myricetin may interact with some surface glycoproteins of hsv. the results of darts assay and gd binding assay indicated that myricetin may interact with virus gd protein rather than cellular receptors of hsv to block virus binding and entry (fig. ) . the data from spr assay verified that myricetin can truly bind to hsv- gd protein with high affinity (kd ≈ . nm) (fig. e) . the molecular docking analysis indicated that myricetin may interact with gln , leu , glu and asn of gd protein through hydrogen bond interactions ( fig. g and h) . furthermore, myricetin treatment inhibited the activation of egfr, pi k and akt proteins in hsv infected or gd treated non-infected cells, and reduced the mrna levels of virus proteins in hsv-infected cells (fig. ) . considered that egfr/pi k/akt pathway is required for efficient hsv replication (tiwari and shukla, ) , we suppose that myricetin may interact with virus gd protein to interfere with the activation of egfr/pi k/akt pathway so as to further inhibit subsequent replication of hsv. the mouse model of hsv intranasal infection has been used for studying hsv induced pneumonia and encephalitis (gamba et al., ) . in this study, intraperitoneal therapy of hsv- -infected mice with myricetin markedly improved their survival and inhibited hsv multiplication in both lungs and spinal cord (fig. ) . moreover, the histopathological analysis indicated that myricetin treatment also significantly attenuated the pneumonia and encephalitis symptoms in hsv-infected mice, comparable to the effects of acyclovir. in contrast to acyclovir, myricetin can block hsv binding and entry process with low toxicity, suggesting that it may be used for therapy and prophylaxis of hsv infection. however, oral administration of myricetin as an antiviral may be unlikely to be successful because of its rapid metabolism. thus, myricetin may be used for prevention and treatment of herpes simplex pneumonia and encephalitis by intravenous administration in the future. in summary, myricetin possessed anti-hsv activities both in vitro and in vivo with low toxicity. myricetin may block hsv infection through direct interaction with virus gd protein to interfering with virus adsorption and membrane fusion. in addition, cellular egfr/ pi k/akt pathway was also involved in the anti-hsv actions of myricetin. thus, myricetin merits further investigation as a novel anti-hsv agent targeting both virus gd protein and host egfr/pi k/akt pathway. further studies of the anti-hsv effects of myricetin against clinical strains especially the acyclovir-resistant strains will be required to advance it for drug development. nevertheless, myricetin has the potential to be developed into a novel antiviral injection for therapy of herpes simplex encephalitis and pneumonia in the future. none declared. the immune response to ocular herpes simplex virus type infection antimicrobial activity of flavonoids antiviral effects of abma against herpes simplex virus type in vitro and in vivo neuroprotection by flavonoids. braz intranasal challenge of mice with herpes simplex virus: an experimental model for evaluation of the efficacy of antiviral drugs a novel glycoprotein d-specific monoclonal antibody neutralizes herpes simplex virus the epidermal growth factor receptor (egfr) promotes uptake of influenza a viruses (iav) into host cells human herpes simplex virus infections: epidemiology, pathogenesis, symptomatology, diagnosis, and management early inhibition of nitric oxide production increases hsv- intranasal infection the potential 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dextran derivatives anti-hiv- activity of flavonoid myricetin on hiv- infection in a dual-chamber in vitro model dietary flavonoids: bioavailability, metabolic effects, and safety a review of the mechanisms and effectiveness of dietary polyphenols in reducing oxidative stress and thrombotic risk myricetin: a dietary molecule with diverse biological activities age-specific prevalence of infection with herpes simplex virus types and : a global review phloroglucinol glycosides from the fresh fruits of eucalyptus maideni role for -o-sulfated heparan sulfate as the receptor for herpes simplex virus type entry into primary human corneal fibroblasts phosphoinositide kinase signalling may affect multiple steps during herpes simplex virus type- entry boronic acid modifications enhance the anti-influenza a virus activities of novel quindoline derivatives identification of myricetin and scutellarein as novel chemical inhibitors of the sars coronavirus helicase, nsp this work was supported by national natural science foundation of china ( , , ), nsfc-shandong joint fund (u , u ), shandong provincial natural science foundation (zr mh ), and qingdao marine biomedical science and technology innovation center ( -cxzx - - ). supplementary data to this article can be found online at https:// doi.org/ . /j.antiviral. . . key: cord- - eqtog authors: pavel, shaikh terkis islam; yetiskin, hazel; aydin, gunsu; holyavkin, can; uygut, muhammet ali; dursun, zehra bestepe; celik, İlhami; cevik, ceren; ozdarendeli, aykut title: isolation and characterization of severe acute respiratory syndrome coronavirus in turkey date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: eqtog coronavirus disease (covid- ) caused by severe acute respiratory syndrome coronavirus (sars-cov- ) and associated with severe respiratory illness emerged in wuhan, china, in late . the virus has been able to spread promptly across all continents in the world. the current pandemic has posed a great threat to public health concern and safety. currently, there are no specific treatments or licensed vaccines available for covid- . we isolated sars-cov- from the nasopharyngeal sample of a patient in turkey with confirmed covid- . we determined that the vero e and ma- cell lines are suitable for supporting sars-cov- that supports viral replication, development of cytopathic effect (cpe) and subsequent cell death. phylogenetic analyses of the whole genome sequences showed that the hcov- /turkey/eragem- / strain clustered with the strains primarily from australia, canada, england, iran and kuwait and that the cases in the nearby clusters were reported to have travel history to iran and to share the common unique nucleotide substitutions. coronaviruses (covs) are members of the family coronaviridae, which consists of a group of enveloped, positive-sense, single-stranded rna viruses [ ] . transcription of coronaviruses requires a polymerase template switch, characterized as a discontinuous process unique among rna viruses [ ] [ ] [ ] . based on the difference in protein sequences, covs are classified into four genera, alpha-cov, beta-cov, gamma-cov, and delta-cov [ , , ] . there are hundreds of coronaviruses are circulating broadly among mammals and birds that cause respiratory, enteric, hepatic, and neurologic diseases [ , , ] . until recently, six coronavirus species have been known to cause disease in humans. the e, oc , nl and hku viruses are prevalent and cause mild illness, such as the common cold [ , ] . however, the other two viruses have been considered highly pathogenic in humans, and cause the diseases sars (severe acute respiratory syndrome), which resulted from an outbreak in and disappeared by , and mers (middle east respiratory syndrome), which emerged in and continues to circulate in the middle east [ ] [ ] [ ] [ ] [ ] . at the end of , severe pneumonia cases of unknown etiology were reported in wuhan, a city in the hubei province of china [ ] [ ] . sequencing analysis revealed that this unidentified pneumonia was considered to be caused by a novel coronavirus [ , ] . the world health organization (who) termed the disease as coronavirus disease- (covid- ) on february , [ ] . on the same day, the international committee on taxonomy of viruses (ictv) named this novel coronavirus as severe acute respiratory syndrome coronavirus (sars-cov- ). sars-cov- has become the seventh coronavirus that known to infect humans. even though the first cases had a contact history with the huanan seafood market the studies have clearly showed that sars-cov- can be transmitted by person-to-person and frequently cause asymptomatic infections. with transmission of the virus possible before the onset of clinical signs, the covid- outbreak has quickly expanded to worldwide [ ] [ ] [ ] . it was declared a pandemic by the who on march , . as of august , , a total of , , confirmed cases of covid- and , deaths have been reported in more than countries and territories. (https://www.who.int/emergencies/diseases/novelcoronavirus- /situation-reports/). the first case of covid- in turkey was confirmed on march . as of august , , there have been , cases and , deaths (the ministry of health, turkey). in this study, we report the isolation of the hcov- /turkey/eragem- / strain from a patient in turkey with confirmed covid- . the whole genomic sequence and replication characteristics of the hcov- /turkey/eragem- / strain are described. this is the first known report of the isolation and characterization of sars-cov- from a human clinical sample in turkey. the successful isolation and characterization of the virus will be essential for continued investigations of sars-cov- pathogenicity and will provide valuable information for vaccine design and drug target. this study protocol was approved by the kayseri training and research hospital ethics committee ( - -/ ), which allowed sampling for diagnostic and surveillance purposes. a written informed consent was obtained from the patient for being included in the study. all cell lines used in this study purchased from atcc cell culture company. african monkey green kidney cells (vero e , atcc crl- ), rhesus monkey kidney cells (ma- , atcc crl- ), human adrenal carcinoma cells (sw- , atcc ccl- ) and human cervical adenocarcinoma cells (hela, atcc ccl- ) were cultured in dulbecco's modified eagle's medium (dmem) (sigma, germany) supplemented with % heat-inactivated fetal bovine serum (fbs) (gibco, usa), mm l-glutamine, u/ml penicillin, μg/ml streptomycin (biological industries, usa). all cell lines tested were found to be free of mycoplasma using the ez-pcr mycoplasma detection kit (biological industries, usa). a patient was admitted to the kayseri city training and research hospital on march , due to respiratory symptoms. the patient's nasopharyngeal sample was obtained by using a utm™ kit containing ml of viral transport media (copan diagnostics, usa) on day of his illness. the diluted sample was inoculated onto monolayers of vero e cells and gently agitated at ˚c for h. consequently, dmem with % fbs was added and the infected cells were monitored for the appearance of cytopathic effect (cpe). all handling of the virus was conducted in a biosafety level enhanced facility (bsl- ). viral rna was isolated from μl of the infected culture supernatant using the qiaamp viral rna mini kit (qiagen, germany) according to the manufacturer's recommendations. the viral rna was reverse transcribed using the moloney murine leukemia virus reverse transcriptase (m-mlv rt) (thermo scientific, usa) using random hexamers according to the manufacturer's recommendations. the reaction mixtures were incubated for min at ˚c, and the reaction was stopped by heating the mixture at ˚c for min and chilling it on ice. the primers used in pcr reactions were designed according to the sequences published by the centers for disease control and prevention (cdc) [ ] . the pcr was conducted in a μl reaction mixture containing μl of cdna template, mm tris-hcl, mm kcl, . mm mgcl , -ncov_n forward primer ( -gaccccaaaatcagcgaaat- ), -ncov_n reverse primer ( -tgtagcacg att gcagcattg - ), u of taq polymerase (thermo scientific, usa), and . mm dntps. the cycling conditions were ˚c for min, followed by cycles of ˚c for sec, ˚c for sec, and ˚c for min with a final extension at ˚c for min. the pcr products were visualized by ethidium bromide staining after % agarose gel electrophoresis. the pcr reactions were also set up with two different combinations of the primers under the same conditions as described above. the primers used in the pcr reactions were -ncov_n forward primer ( -gaccccaaaatcagc gaaat- ), -ncov_n reverse primer ( '-gcgcgacattccgaagaa- ') and -ncov_n forward primer ( '-gggagccttgaatacaccaaaa- '), and -ncov n reverse primer ( '-gcg cgacattccgaagaa- '), respectively. twenty-four-well plates were seeded with vero e cells and incubated at ˚c with % co . the monolayer was inoculated with -fold serially dilutions of the virus. after incubation for h at ˚c with shaking, the monolayer was overlaid with . ml overlay medium containing . % low melting agarose (sigma, germany). after incubation at ˚c for days, the cells were fixed with % formalin (v/v) for min at room temperature. the agarose overlay was discarded, and the plaques were visualized by staining the monolayer with % crystal violet (w/v) in % ethanol (v/v). we cultured to sars-cov- passage ( : dilution) in vero e cells to make virus the passage virus stock. the sars-cov- virus lysate was then harvested at h post-infection and the supernatants were collected, clarified, and stored at - ˚c. to determine the titer of the passage virus a focus forming assay (ffa) was performed as described previously [ ] . briefly, vero e cells were seeded on well-plates and incubated at ˚c with % co for overnight. the cell monolayers were inoculated with -fold serial dilutions virus at nd passage. the diluted samples were added in triplicate to confluent vero cell monolayers. after absorption for h at ˚c, the supernatants were removed and the cells were washed with pbs. the cell monolayers were overlaid with virus medium containing % cmc (carboxymethyl cellulose) then incubated at ˚c with % co for h. after fixation with % neutral buffered formaldehyde at room temperature for min, the cells were permeabilized with . % triton x- in pbs for min with gentle rocking and blocked with % skim milk in pbs. the wells were then incubated with a human antibody to sars-cov- nucleocapsid protein ( : ) (genscript; hc ) for h in tbst ( mm tris-hcl ph . , . m nacl, % tween ) at ˚c and washed times with tbst. the cells were incubated for h with goat anti-human igg conjugated to fluorescein isothiocyanate (fitch) (southern biotech, usa) and diluted : in tbst and then washed three times with tbst and once with distilled water. the antibody-labeled cells were detected and analyzed by immunofluorescence microscopy (leica, uk). the fluorescent foci in each well were counted, and the virus titers were calculated and expressed as fluorescent focus units (ffu) per ml as described previously [ ] . to obtain the virus passage virus stock, the vero e cells were infected with the virus passage virus at an moi of . , and the viral lysate were was harvested at h post-infection and the supernatants were collected. subsequently, the virus passage virus was generated in vero e- cells infected with virus passage virus at an moi of . . cell lysates were harvested in laemmli sodium dodecyl sulfate-polyacrylamide (sds) gel electrophoresis sample buffer containing % sds and % β-mercaptoethanol. the lysates were boiled and loaded onto a polyacrylamide gel. the samples were separated on % resolving and % stacking sds-page gels in a mini electrophoresis unit (bio-rad, usa) at v for h. the proteins were transferred onto a nitrocellulose membrane (millipore, usa) under wet conditions using a trans-blot apparatus (bio-rad, usa). after blocking with % skimmed milk, the membrane was incubated either with a rabbit polyclonal to sars-cov- spike glycoprotein ( / ) (abcam; ab ) or a human antibody to the sars-cov- nucleocapsid protein ( : ) (genscript; hc ) followed by a goat anti-rabbit horseradish peroxidase (hrp)-conjugated antibody ( : dilution, invitrogen; usa) and a goat anti-human horseradish peroxidase (hrp)-conjugated antibody ( : dilution, invitrogen; usa), respectively. b-actin was used as a loading control in western blot. the membrane was reacted with the ecl substrate solution (pierce ecl, usa). the membrane was exposed to an autoradiograph film (kodak x-omat, sigma germany), and was developed using a kodak developer (x-omat a, sigma germany). vero e- and ma- cells cultured in -well-plates were infected with an moi at an . (passage virus). the cultures were harvested by scraping cell monolayers from at different time points ( , , , , and h) and stored at - ˚c. the vero e- cells were then inoculated with -fold serial dilutions of the samples in triplicate per dilution. the viral inoculum was removed. the cell monolayers were overlaid with virus medium containing % cmc. the cells were fixed with % neutral buffered formaldehyde after infection of h at room temperature for min, and permeabilized with triton x- . the wells were then incubated with a human antibodt to the sars-cov- nucleocapsid protein ( : ) (genscript; hc ) for h in tbst at ˚c and washed times with tbst. the cells were incubated for h with goat anti-human igg conjugated to fluorescein isothiocyanate (fitch) (southern biotech, usa) and diluted : in tbst and then washed three times with tbst and once with distilled water. antibody-labeled cells were detected and analyzed by immunofluorescence microscopy (leica, uk). the fluorescent foci in each well were counted, and the virus titers were calculated and expressed as fluorescent focus units per ml. for whole genome sequencing of hcov- /turkey/eragem- / , vero e cells infected with the virus were used for rna extraction. the rna was extracted by using the qiaamp viral rna mini kit (qiagen, germany). the viral rna was reverse transcribed by m-mlv rt using random hexamers according to the manufacturer's recommendations. the dna amplicons the from full genome amplification [ ] were quantified using the quant-it dsdna hs assay kit (invitrogen, usa) and pooled in equal concentrations. the libraries were prepared using pooled amplicons with nextera dna flex library prep kit (illumina, san diego, ca) and sequenced on an illumina nextseq (illumina, usa) platform with a x cycle kit (gen era diagnostics inc., turkey). the quality of the raw data was evaluated by fastqc v. . . (babraham bioinformatics) and low-quality bases, primers and remnant adapters were trimmed using trimmomatic v. . [ ] . the reads were aligned to the previously assembled sequence of the sars-cov- genome (genbank accession: mn . ) using the burrows-wheeler aligner v. . . with the mem algorithm [ ] . the variants were called by using genome analysis toolkit (gatk) v. . . with the haplotypecaller algorithm [ ] and were manually inspected in genomebrowse v . . (goldenhelix). the variants that had low quality and a low variant fraction (%< ) were filtered. the filtered variants and reference sars-cov- genome were used to generate the consensus sequence using bcftools v . [ ] . for multiple sequence alignment, complete (> , bp) and high-coverage genomes (n = ) were used from the gisaid database. the gisaid strain genomes including the genome of our strain were aligned using the mafft v . tool [ ] . phylogenetic analysis of the alignment was performed using the iq-tree v. . . with a general time-reversible (gtr) model [ ] . the whole genome sequence was submitted to genbank (id:mt . ) and gisaid (id: epi_isl_ ) and the raw data deposited on sra (samn ). graphpad prism software (graphpad, usa) was used to perform all statistical analysis and graphics. mann-whitney u test was used to find significant differences between viral passages. the significance level was set as a p value of less than . where � p< . . after h of incubation, very little but visible cpe was detected in virus passage virus ( fig b) . the time for the onset of cpe was typically h post-infection ( fig c) and major cpe was observed within h post-infection ( fig d) . the cells showed some morphological changes such as cell rounding, detachment/floating and degeneration whereas no such changes were observed in the uninfected cells (fig a, b, c and d) . as we observed cpe in the infected monolayers, rt-pcr was used as a confirmatory assay. the primer set from cdc [ ] targeting the nucleocapsid protein gene (np) of sars-cov- was used for the pcr reactions. a pcr product size of bp was amplified with the -ncov_n forward and -ncov_n reverse primers (fig e, lane ) . we amplified two pcr products, with sizes of bp and bp, with the -ncov_n forward, and -ncov_n reverse primers and with the -ncov_n forward and -ncov_n reverse primers, as in shown fig e lane and fig e lane , respectively. we also performed to plaque assay to purify of sars-cov- for subsequent use in further experiments. representative sars-cov- plaques in the vero e cell monolayers infected with sars-cov- are shown in fig f and g . taken together, these results suggest that a sars-cov- strain named hcov- /turkey/eragem- / was successfully isolated from the nasopharyngeal sample of a patient in turkey with confirmed covid- . we cultured to sars-cov- passage in vero e cells to make the virus passage virus stock. subsequently, the passage virus stock was passaged two more times in vero e cells. the virus stocks were quantified by using ffa (fig ) . we determined that the titer of the passage virus was . x ffu/ml, while the titers of the passage and passage viruses were . x ffu/ml and . x ffu/ml, respectively (fig ) . these results indicated that propagation of the hcov- /turkey/eragem- / strain in vero e cells led to an increasing in the viral titers in each passage. in addition to vero e cells, we examined the susceptibility of ma- , sw- and hela cell lines to infection by sars-cov- . all cell lines were infected with an moi of . (virus passage virus) and monitored for cpe until h post-infection. only the ma- cell line developed cpe. in abnormal areas, small clusters of rounded cells, cell detachment and degeneration were observed (fig ) . similar to vero e cells infected with sars-cov- , cpe formation in the ma-infected cells began at h post-infection ( fig b) and increased at h post-infection (fig c) . the complete cpe was observed within h post-infection (fig d) . to confirm the results of the susceptibility of the ma- , sw- and hela cell lines to infection by sars-cov- , all cell lines were infected with an moi of . (passage virus) and incubated at h post-infection. an immunofluorescence assay (ifa) confirmed that the vero isolation of sars-cov- in turkey e and ma- cell lines supported the replication of sars-cov- . in contrast, sars-cov- did not replicate in sw- and hela cells. (fig ) . to expand these observations, we examined the expression of the sars-cov- proteins. all cell lines were infected with an moi of . (passage virus). cell lysates from infected cell lines were harvested at h post-infection and were probed either with the rabbit polyclonal antibody to sars-cov- spike glycoprotein or with a human antibody to the sars-cov- nucleocapsid protein. sars-cov- spike protein (s) expression was detected in vero e and ma- cell lines that supported sars-cov- replication (fig a) . the vero e and ma- cell lines also showed a sars-cov- nucleoprotein (np) band, as shown in fig b. consistent with the ifa results (fig ) , viral antigen expression was not detectable in the nonsusceptible sw- and hela cell lines (fig ) . overall, these results showed that the vero e and ma cell lines can be efficiently infected by sars-cov- . to assess the replication kinetics, vero e and ma- cells were infected with at an moi of . , and the supernatants were harvested at different time points ( , , , , and h post-infection). the vero e and ma- cell monolayers were then inoculated with -fold serial dilutions of the samples. viral titers of the samples were determined by ffa (fig a) . the growth kinetics study showed that sars-cov- replicated rapidly and efficiently and could be detected within h post-infection in vero e and ma- cells (fig a and b ). the western blot assay was performed to examine the production of viral proteins using a rabbit polyclonal antibody to the sars-cov- spike glycoprotein (s) ( / ) (abcam; ab ) and a human antibody to the sars-cov- nucleocapsid protein (np) ( : ) (genscript; hc ). the membrane was reacted with the ecl substrate solution (pierce ecl, usa). the membrane was exposed to an autoradiograph film (kodak x-omat, sigma germany), and was developed using a kodak developer (x-omat a, sigma germany). the arrows indicate that the bands at approximately kda ( fig a) and kda (fig b) sars-cov- replicated in vero e and ma- cells with similar kinetics and achieved similar peak titers of . xlog ffu/ml and . xlog ffu/ml at h of post-infection, respectively ( fig a) . however, the viral titers decreased after h infection. at h post-infection, the titers from the samples vero e and ma- infected with sars-cov- were . xlog ffu/ml and xlog ffu/ml, respectively (fig a) . the sequencing produced approximately . m paired reads ( bp x ), of which . % of the reads were mapped to the reference genome. after alignment, . % of the genome was covered with a x sequencing depth on average. the whole-genome sequencing of hcov- /turkey/eragem- / revealed that the strain had variants, compared to the mn . reference genome. the detected non-synonymous, synonymous, and utr variants are listed in table . the variants were found to correspond to the genomic positions and (orf ab gene), (s gene), (np gene), (orf gene) and ( ' utr) . the mutations at and are non-synonymous, leading to a change from valine to isoleucine in genes orf ab and s (table ) . the phylogenetic analysis showed that hcov- /turkey/eragem- / was located outside of the main clades (s, g, and v) and clustered with sars-cov- isolates from australia, canada, england and kuwait (fig ) . this geographically dispersed cluster is known to be branched in the early period of the epidemic and genetically clustered very closely together. our strain shared three distinct mutations (g a, t c, and g t) with all members of this cluster, and those mutations were not observed in other known clusters. according to published case reports and gisaid metadata entries, at least sars-cov- samples in the nearby clusters had a recent travel history to iran [ ] . those samples in nearby clades include several cases from pakistan, kuwait, canada, and norway (fig ) . the genome sequences of all those cases with a history of travel to iran share three nucleotide substitutions (g a, t c, and g t) in the sars-cov- genome, which were also found in the hcov- / turkey/eragem- / strain. the gisaid had only one full genome sequence of sars-cov- from iran, which also included the two key mutations (g a and g t). in addition, analysis of the np partial gene sequences of the iranian samples in gisaid showed that all of the iranian partial sequences (n = ) also contain the t c, which is another key single nucleotide polymorphism (snp) of this clade. our strain also contained g a, a non-synonymous mutation in the s gene, which was not observed in any other full genome sequences in gisaid. the mutation leads to a val to ile change at the nd position, which is located at one of the inner coil motifs of the s protein. another mutation in the orf gene (c t) is also quite rare and found only in two cases in australian samples. this study generated the second whole genome sequence of a sars-cov- strains in turkey. the first sequence was generated in march and deposited in gisaid (epi_isl_ ). the first sequence was not included in the phylogenetic analysis because of its high number of unique mutations ( . %). however, the sequence comparison showed that both sequences carry key iranian cluster mutations including g a, t c, and g t. the g t, g a, and c t nucleotide changes in our strain were not found in the first sequenced strain in turkey. as of april , a total of sars-cov- sequences have been submitted to gisaid samples from turkey. two of those sequences (turkey/hgsm/ / and turkey/hgsm/ / ) were located in the same cluster and share cluster-specific variants (g a, t c, g t). the rest of the submitted strains clustered with several other european clades. sars-cov- is an emerging coronavirus that is highly infectious and efficiently transmitted through droplets and close contact. the virus has been able to spread promptly to several countries throughout the world [ ] [ ] [ ] [ ] [ ] . the sars-cov- pandemic is likely to have serious effects on not only people's health but also societies, health system worldwide and the global isolation of sars-cov- in turkey economy [ ] [ ] [ ] . in the current outbreak situation, it is crucial to isolate of the causative virus for vaccine strain production, initial characterization of antiviral candidates and evaluation of diagnostic tools. sars-cov- was first isolated by using human airway epithelial cells on january , [ , ] . following to the first isolation of sars-cov- in china, several groups have also isolated sars-cov- by using the vero cell line [ ] [ ] [ ] [ ] . in this study, we isolated the sars-cov- from the nasopharyngeal sample of a patient in turkey with confirmed covid- . isolation of sars-cov- was successfully achieved in vero e cells in the absence of trypsin. sars-cov- caused morphological changes such as rounding, detachment/floating and degeneration (fig b, c and d ). it is essential to define different target cells for sars-cov- for further studies on virushost interactions. chu and colleagues identified different cell lines in which both sars-cov and sars-cov- replicated efficiently, but the cytopathic effects were only seen in the nonhuman primate kidney cell lines veroe and frhk [ ] . recently, a study showed that the vero e and vero ccl cell lines were infected with sars-cov- and exhibited to sars--cov- specific cpe. these authors found that huh . and t cells showed only modest viral replication but no cpe was observed, suggesting that both vero cell types support amplification and replication of sars-cov- but that vero e cells are more suitable for amplification and quantification [ ] . in this study, we assessed the susceptibility of vero e , ma- , sw- and hela cell lines to infection by sars-cov- . we determined that the vero e and ma- cell lines were permissive for sars-cov- infection. initial cpe formation in vero e and ma- cell lines infected with sars-cov- was observed as early as at h postinfection (figs b and b, respectively) . immunoblotting analysis also confirmed that only vero e and ma- cell lines infected with sars-cov- showed the expression of the virus specific proteins expression (fig ) . in addition to vero e cells, the ma cell line pertaining to its suitability for sars-cov- proliferation and facilitate further study of sars-cov- . our results are in agreement with the previous reports showing that sars-cov replicated efficiently and caused cpe in vero and ma- cell lines [ ] [ ] [ ] [ ] . in order to evaluate the viral growth kinetics of sars-cov- , the vero e and ma- cell lines were infected with at an moi of . . viral replication was assessed at different time points ( , , , , and h post-infection). we used ffa for all virus titration experiments in this study, since ffa is independent of cell death. sars-cov- replicated with a similar kinetics in vero e and ma- cells, but the ma- cells supported replication of sars-cov- slightly less well than the vero e cells (fig a) . viral replication could be detected at h post-infection, and continued to increase gradually, peaking at h post-infection (fig a and b ). the decline in virus titer at h post-infection which might be due to death of the infected cells or to the cell lysis ( fig a) . our data are in agreement with that of harcourt et.al., in which sars-cov- replicated rapidly in vero cells after an initial eclipse phase and increased gradually, peaking at h post-infection [ ] . lastly, we have compared the whole genome of hcov- /turkey/eragem- / with a dataset of available sars-cov- complete genomes from different countries retrieved from gisaid. the hcov- /turkey/eragem- / strain was closely clustered with other strains primarily from australia, canada, england, iran, and kuwait. the reason for the high fraction of australian cases in this cluster was possibly due to the high number of submitted sequences, as australia was the third country that had the most sequences submitted to gisaid by april . the cases in the nearby clusters were reported to have a travel history to iran and shared the common unique nucleotide substitutions, g a, t c, and g t, which were also found in our strain and partially in several iranian cases [ ] . the common key mutations and similar travel histories of the closely clustered cases may indicate possible links between our case and the iranian epidemic. interestingly, hcov- /turkey/ eragem- / has the g a mutation on the s gene, which is not found in any other full genome sequences in gisaid, including the australian cluster. the lack of some variants (g t, g a, and c t) in the previously sequenced turkey strains may also show multiple introductions of strains in the early stages of the epidemic. in this regard, the potential limitation of this finding is the limited number of sequences available for analysis. further studies with more sequences are needed to determine the distribution of this variant in turkey and tracing the origin of this strain. we have describe the successful isolation of sars-cov- from a patient in turkey with confirmed covid- . we determined that the vero e and ma- cell lines might be a good choice of cell culture model for sars-cov- that supports viral replication kinetics, development of cpe and subsequent cell death. we also showed that hcov- /turkey/eragem- / was closely clustered with other strains primarily from australia, canada, england, iran, and kuwait and that the cases in the nearby clusters were reported to have a travel history to iran and to share the common unique nucleotide substitutions. 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pneumonia "covid- a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study centers for disease control and prevention home page application of the pseudo-plaque assay for detection and titration of crimean-congo hemorrhagic fever virus a new coronavirus associated with human respiratory disease in china trimmomatic: a flexible trimmer for illumina sequence data fast and accurate short read alignment with burrows-wheeler transform a framework for variation discovery and genotyping using next-generation dna sequencing data bcftools/roh: a hidden markov model approach for detecting autozygosity from next-generation sequencing data mafft multiple sequence alignment software version : improvements in performance and usability iq-tree: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies an emergent clade of sars-cov- linked to returned travellers from iran an interactive web-based dashboard to track covid- in real time characteristics and outcomes of critically ill patients with covid- in washington state host and infectivity prediction of wuhan novel coronavirus using deep learning algorithm first case of novel coronavirus in the united states the novel zoonotic covid- pandemic: an expected global health concern economic impacts of wuhan -ncov on china and the world the socio-economic implications of the coronavirus and covid- pandemic: a review multidisciplinary research priorities for the covid- pandemic: a call for action for mental health science. lancet psychiatry who. novel coronavirus( -ncov) situation report- virus isolation from the first patient with sars-cov- in korea serological and molecular findings during sars-cov- infection: the first case study in finland severe acute respiratory syndrome coronavirus from patient with novel coronavirus disease, united states. emerg infect dis inhibition of sars-cov- infections in engineered human tissues using clinical-grade soluble human ace . cell comparative tropism, replication kinetics, and cell damage profiling of sars-cov- and sars-cov and implications for clinical manifestations, transmissibility, and laboratory studies of covid- : an observational study sars-associated coronavirus replication in cell lines severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis exogenous ace expression allows refractory cell lines to support severe acute respiratory syndrome coronavirus replication our gratitude is expressed towards the staff at the infectious diseases ward at the kayseri city training and research hospital for providing us with the fresh material for this study. conceptualization: aykut ozdarendeli. key: cord- -rpv oy authors: park, jung-eun; cruz, deu john m.; shin, hyun-jin title: receptor-bound porcine epidemic diarrhea virus spike protein cleaved by trypsin induces membrane fusion date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: rpv oy porcine epidemic diarrhea virus (pedv) infection in vero cells is facilitated by trypsin through an undefined mechanism. the present study describes the mode of action of trypsin in enhancing pedv infection in vero cells during different stage of the virus life cycle. during the viral entry stage, trypsin increased the penetration of vero-cell-attached pedv by approximately twofold. however, trypsin treatment of viruses before receptor binding did not enhance infectivity, indicating that receptor binding is essentially required for trypsin-mediated entry upon pedv infection. trypsin treatment during the budding stage of virus infection induces an obvious cytopathic effect in infected cells. furthermore, we also show that the pedv spike (s) glycoprotein is cleaved by trypsin in virions that are bound to the receptor, but not in free virions. these findings indicate that trypsin affects only cell-attached pedv and increases infectivity and syncytium formation in pedv-infected vero cells by cleavage of the pedv s protein. these findings strongly suggest that the pedv s protein may undergo a conformational change after receptor binding and cleavage by exogenous trypsin, which induces membrane fusion. porcine epidemic diarrhea virus (pedv), a member of the family coronaviridae, is an economically important pathogen of swine. pedv causes acute watery diarrhea, resulting in approximately % mortality among suckling piglets and reduced weight among fattening pigs [ ] . although the structural and pathological properties of pedv are similar to those of other group coronavirus, including human coronavirus e (hcov- e), transmissible gastroenteritis virus (tgev) and feline infectious peritonitis virus, many biological issues, such as the role of trypsin in infection, remain unresolved [ , , ] . the first successful propagation of pedv in cell culture was done by supplementing the vero cell culture medium with trypsin [ ] . the addition of trypsin was shown to induce fusion of the infected vero cells, resulting in the formation of multiple syncytia, and produced a significant increase in virus titer after several passages. soon afterwards, several other groups performed pedv infection in vitro using the same conditions and reported similar findings [ , ] . on the other hand, another study reported the successful propagation of the p- v strain in porcine enterocyte cell lines without trypsin supplementation of the medium, suggesting that the proteolytic processing of pedv in enterocytes may have occurred during maturation or prior to virus release [ ] . the spike (s) glycoprotein is the dominant surface protein in coronaviruses. the protein is responsible for virus attachment and fusion. the requirement of proteinase-cleaved s glycoprotein has been reported for almost all group and coronavirus. for example, infection by severe acute respiratory syndrome coronavirus (sars-cov) and murine hepatitis virus strain (mhv- ) requires proteolytic cleavage in their target cells, which is mediated by trypsin-like proteases [ , , ] . the situation for group coronavirus is unclear. recently, the s protein of the group coronavirus hcov- e was reported to be cleaved by treatment with cathepsin l and trypsin, which prompts the fusion of the viral envelope and the cell membrane, similar to sars-cov [ ] . the present study reports the putative role of trypsin in cell-adapted pedv infection of vero cells. trypsin treatment was performed in the early and late stages of viral infection, and its influence on viral titer and syncytium formation was examined. furthermore, the effect of trypsin on the s protein was compared in free and receptor-bound virions. the results suggest that trypsin activity is involved mainly with receptor-bound s protein of pedv, leading to the conclusion that the effect of trypsin on pedv is similar to that of the group coronaviruses, sars-cov and mhv. african green monkey kidney cells (vero, ccl- ) were prepared in minimum essential medium (mem, gibco) supplemented with % fetal bovine serum (fbs, gibco). the cell-adapted strain of the korean pedv isolate, kpedv- , was propagated as described elsewhere [ ] , with some modifications. briefly, vero cells were inoculated with kpedv- at a multiplicity of infection c and cultured in serum-free mem at °c, % co for - h. the supernatant was harvested and then clarified by centrifugation at , g for min at °c. concentration and partial purification were performed by ultracentrifugation under a % sucrose cushion at , rpm for . h. the pellet was resuspended in mm phosphate-buffered saline (pbs, ph . ) and stored at - °c. mouse polyclonal antibodies against pedv were generated by immunizing -week-old female balb/c mice (samtako) intraperitoneally with focus-forming units (ffu) of purified kpedv- emulsified in an equal volume of complete freund's adjuvant (sigma-aldrich) on day and incomplete freund's adjuvant (sigma-aldrich) on days , and . whole blood was collected from the retro-orbital sinus on days and and centrifuged at g for min to separate the sera. cultured vero cells were inoculated with kpedv- as described above and allowed to adsorb for h at °c. the vero cells were washed twice with pbs and cultured in serum-and trypsin-free mem or mem containing trypsin ( lg/ml, sigma-aldrich). at , , , and h postinoculation (hpi), culture supernatants were collected for titration in a focus-formation assay, and cells were fixed with % formaldehyde in pbs for min and permeabilized with % np- (sigma-aldrich) in pbs, followed by immunocytochemistry using mouse anti-pedv polyclonal sera [ ] . clusters of infected cells staining dark gray were counted under an inverted microscope and reported as ffu. trypsin treatment at various stages of virus infection cells or viruses were treated with trypsin at various stages of virus infection as described in fig. . to investigate the effects of proteolytic cleavage of the surface protein of vero cells and free virions by trypsin, vero cells or kpedv- were pre-treated with trypsin prior to infection. trypsin treatment was performed for min at °c prior to inoculation, and enzyme activity was neutralized with lg/ml aprotinin (sigma-aldrich). trypsin-pretreated vero cells were inoculated with kpedv- , and untreated vero cells were inoculated with trypsin-pretreated kpedv- . after a -h incubation to allow adsorption, cells were washed three times with pbs and then cultured in serum-free mem without trypsin for h. in another experiment, trypsin treatment was carried out during the virus adsorption stage to determine whether trypsin is involved in the entry of kpedv- . vero cells were inoculated with kpedv- in the presence of various concentrations of trypsin ( , , , and lg/ml) during the adsorption period and were cultured in serum-free mem at °c for h. to investigate the effect of trypsin on the budding stage of pedv infection, kpedv- -infected vero cells were prepared by inoculating them with purified kpedv- and then cultured in mem for h. prior to trypsin treatment, the cell monolayer was washed three times with pbs to remove residual fbs and released virions, prior to treatment with trypsin for min. after neutralization of trypsin by the addition of aprotinin, cells were cultured for an additional h. the culture supernatants were harvested for virus titration, and cells were fixed for immunocytochemistry at the indicated times. virus-infected cells were detected by probing with mouse anti-pedv polyclonal antisera and biotinylated rabbit antimouse igg and visualized by treatment with streptavidinbiotinylated horseradish peroxidase (vector labs) followed by , '-diaminobenzidine tetrahydrochloride dihydrate (dab, vector labs). all specimens were observed under an inverted microscope. ultrapurified kpedv- was treated with various concentrations of trypsin at room temperature (rt) for min and then analyzed by western blotting. vero cells were infected with kpedv- in the absence of trypsin for h, and kpedv -infected vero cells then were harvested and treated with trypsin for min at rt. mock-infected trypsin-treated vero cells were used as a negative control. samples for western blot analysis were treated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) sample buffer and electrophoresed in an % sds-page gel system. the separated proteins were electrically transferred onto a polyvinyl difluoride membrane (amersham bioscience). the antibodies used in this study were mouse anti-pedv polyclonal antibodies against pedv s protein, and monoclonal anti-b-actin-peroxidase (sigma-aldrich). the bands were visualized using supersignal west dura (pierce) with las- plus (fujifilm). statistical analysis was performed using spss, version . , for windows. correlation coefficients were calculated using pearson's correlation coefficient. error bars represent the standard deviations from at least three replicates. trypsin is not essential for pedv infection pedv propagation in vero cells results in low infection rates, even after subsequent passages in the absence of trypsin supplementation [ ] . however, with the addition of trypsin, virus adaptation to vero cells increased, and a prominent cytopathic effect (cpe) marked by formation of syncytia was observed in subsequent passages. following this observation, the growth rate of kpedv- in vero cells in the presence or absence of trypsin supplementation was compared. as shown in fig. , detectable levels of progeny virions were observed from hpi in both trypsin-and nontrypsin-supplemented media. at hpi, the titer was higher ( . ffu/ml) in trypsin-supplemented samples than in non-trypsin-supplemented samples ( . ffu/ml). the rate of virus production in trypsin-supplemented cultures was also significantly higher than in trypsin-free cultures (virus titer of . and . ffu/ml at hpi, respectively). even at hpi, the titer in the trypsinfree cultures only reached peak titer levels of . ffu/ml, which was significantly lower than the peak titer attained at hpi in trypsin-supplemented cultures. these results are consistent with the suggestion that trypsin is not absolutely essential for vero-cell-adapted pedv infection, as reported for other group coronaviruses, but the titer increases during infection with trypsin-treated virus. trypsin mediates the penetration of cell-attached pedv to investigate how trypsin enhances pedv infectivity of vero cells, trypsin was added during various stages of infection. trypsin treatment of kpedv- prior to inoculation did not significantly differ from non-trypsin-treated virus after hpi ( . and . ffu/ml, respectively) (fig. ) . this suggests that proteolytic processing of the surface glycoprotein by trypsin prior to receptor binding does not have a significant effect on enhancing infectivity. to determine whether trypsin interaction with vero-cell-surface proteins contributes to enhanced pedv infectivity, vero cells were pre-treated with lg/ml trypsin for min before inoculation. this treatment did not significantly alter virus titer when compared to the untreated cells. interestingly, addition of trypsin immediately after inoculation during the absorption to investigate the mechanism of trypsin in more detail, vero-cell-bound pedv was treated with trypsin. vero cells were inoculated with kpedv- in serum-and trypsin-free media for h and then washed twice to remove un-bound kpedv- . the cell-bound kpedv- was treated with different concentrations of trypsin for min, and the titers of penetrating and produced virus were determined. when the concentration of trypsin in the medium was increased from lg/ml to lg/ml, the number of pedv penetrating the vero cells also increased from ffu/ml to ffu/ml. the enhanced trypsin-mediated penetration during initial infection resulted in an increase in virus titer at hpi, from . ffu/ml to . ffu/ml (fig. ) . these findings are consistent with the notion that trypsin activity during the initial stage of virus infection enhances the efficiency of virus penetration into vero cells, thereby increasing viral infectivity. although the penetration of cell-attached virions was facilitated by trypsin treatment, virions treated with trypsin before cell attachment did not show any difference when compared to the results obtained in the absence of trypsin. based on these findings, it is appropriate to suggest that trypsin might only affect the receptor-bound spike, inducing fusion between the cell membrane and the virus envelope, leading to increased virus penetration. to investigate the role of trypsin on the late stage of infection and syncytium formation, kpedv- -infected vero cells were prepared by inoculation for h. the cells were washed extensively and treated with various concentrations of trypsin for min prior to continuing cell cultivation in fresh serum-and trypsin-free medium. kpedv- -infected vero cells did not show visible signs of syncytium formation in the absence of trypsin, while kpedv- -infected vero cells treated with , and lg/ml trypsin at hpi contained multiple syncytia (fig. a) . without trypsin treatment, the virus titer at hpi was . ffu/ml while kpedv- -infected vero cells treated with , , , and lg/ml trypsin showed virus titers of . , . , . , . and . ffu/ml, respectively (fig. b) . in virus budding stage, trypsin also activated syncytium formation of infected vero cells and consequently increased virus infectivity. newly packaged virions budding from infected vero cells could be activated by trypsin, which caused the infected vero cells to form syncytia. this finding was consistent with the previous results shown in figs. and . the collective results supported the idea that trypsin acts on cell-attached virions, both during virus attachment and during virus release and induces membrane fusion between the host-cell membrane and the virus envelope, and also between host-cell membranes. cleavage of receptor-bound s protein by trypsin the s protein from ultrapurified virions and receptor-bound virions was treated with trypsin and analyzed by western blotting. s protein from both ultrapurified virions and kpedv- -infected vero cells that had not been treated with trypsin was apparent as a species of about kda, which represented the glycosylated native s protein (fig. ) . in ultrapurified virus, only this protein species was detected, even after trypsin treatment, while -kda and -kda proteins, likely trypsin-cleaved s protein, were detected in trypsin-treated kpedv- -infected vero cells (fig. ) . the findings supported the suggestion that pedv s protein has a site that is highly sensitive to trypsin cleavage to produce two fragments, as has been reported for other coronaviruses [ , , ] . however, this fig. enhancement of cellattached kpedv- penetration by trypsin. vero cells were inoculated with kpedv- for h and then washed to remove unbound kpedv- . only cellattached kpedv- was treated with trypsin for min, and penetrated virus (j) and progeny virus were titrated after h (h). increasing the amount of trypsin added during virus adsorption resulted in increased virus penetration into vero cells during initial entry and higher virus titers after hpi cleavage only occurred when the virus was associated with receptor protein. several enterotropic or pneumotropic viruses, such as those belonging to the families orthomyxoviridae and paramyxoviridae, undergo proteolytic cleavage of their surface glycoprotein prior to entry into host cells to facilitate virus penetration by activating the fusion domain of the surface glycoprotein [ , ] . the activated fusion protein undergoes conformational changes that induce fusion of the viral envelope and host membranes [ , , ] . this process usually occurs during the period between virus maturation and virus attachment to the host receptors [ , ] . after the fusion process, the viral core, including the viral genome, is transported into the cytoplasm where uncoating and replication ensue [ ] . in natural infections, the viral surface glycoproteins that are not cleaved during maturation are subsequently cleaved by exogenous proteases secreted from host pancreas, liver and bronchiolar epithelia [ ] . in cell culture, the protease cleavage that is required for virus propagation is carried out by exogenous proteases such as trypsin or pancreatin [ , , ] . these exogenous proteases induce syncytium formation by activating the fusion domain of the viral glycoprotein expressed on the surface of infected cells [ , ] . several studies of different coronaviruses have shown that proteolytic cleavage of the s protein enhances viral infectivity. for mhv, separation of the s and s subunits enhances the fusion activity of the s subunit and increases viral infectivity [ , ] . mutations that alter the furin protease recognition sequence (rxr/kr) located at the junction of the s and s subunits as well as treatment with a peptide furin inhibitor prevent the proteolytic cleavage of the s protein, resulting in reduced cell-cell fusion activity, although viral entry is not significantly affected [ , , ] . conversely, the addition of trypsin to the culture medium can enhance the fusion of mhv-infected cells [ ] . in contrast to mhv, the s protein of sars-cov does not show any evidence of proteolytic maturation to cleaved s and s subunits in mature virions [ ] . instead, proteolytic cleavage of the s protein on the surface of infected cells occurs by exogenous proteases, mediating cell-cell fusion [ , ] . similar to sars-cov, the s protein in most group coronaviruses also does not exhibit cleaved s and s subunits during virus maturation and biogenesis [ ] . several studies on tgev and pedv have used trypsinsupplemented culture media to induce cpe by syncytium formation in st cells and vero cells, respectively [ , ] . furthermore, in the case of pedv, trypsin facilitates successful propagation in vero cells as well as other primate cell lines [ , ] . however, the role of cellular and exogenous proteases on the cell entry of group coronaviruses, particularly pedv, as well as the putative proteolytic cleavage site on the s protein, remains unclear. based on the present results summarized in figs. , and , enhancement of virus penetration and cellcell fusion induced by the addition of trypsin suggests that the pedv s protein may also be cleaved into s and s subunits during the course of infection. electrophoretic examination of purified virions resulted in the detection of the pedv s protein as a monomer of about kda in the absence of trypsin, while the protein was detected as two fragments of kda and kda in the presence of trypsin (fig. ) . it may be that the pedv s protein on native virions adopts a conformation that protects it from various exogenous proteases, but the s protein attached to the host receptor protein may undergo a conformational change that exposes a trypsin cleavage site. previous reports have described the formation of syncytia by pedvinfected cells only upon addition of trypsin in the culture medium, and sequence analysis of the pedv s protein has revealed the absence of the rrx(r/h)r motif, which is associated with cleavage into the s and s subunits [ ] . this suggests that the pedv surface glycoprotein does not undergo proteolytic processing upon maturation and release. conversely, the observation that trypsin can induce temperature for min. pedv s was detected using anti-pedv polyclonal antibodies raised in mice. uncleaved s protein and cleaved s protein are indicated by black and white arrows, respectively cell-cell fusion in pedv-infected cells suggests that proteolytic processing of the s protein by exogenous trypsin may augment viral entry by facilitating fusion of the viral membrane with the host membranes [ , ] . it seems that the timing of the cleavage of the s protein by trypsin is critical for the activation of fusion activity. as shown in fig. , early activation of the s protein before binding to cellular receptors did not enhance viral entry into the host cell, while addition of trypsin shortly after receptor binding increased the efficiency of virus entry. while the s protein in infected cell lysates and receptorbound virions was cleaved into two fragments, the s protein cleavage in pedv was different from that of other coronaviruses, as pedv s protein was only cleaved when associated with its host cell. this implies that cleavage of the s protein by trypsin occurs only when it is bound at the surface of host cells to the host receptor protein, which presumably induces a conformational change in the bound s protein. this conformational change might expose a trypsin cleavage site. cleavage of the s protein could result in membrane fusion. it would be of interest to determine the nature of the conformational change that is involved and the location of the s protein cleavage site. in summary, the present results reveal the role and importance of trypsin in pedv infection of vero cells. trypsin is not essential for pedv infection but enhances its infectivity and cpe formation. trypsin cleaves pedv s protein only when it bound its cell receptor and in the later stages of infection. the association of the s protein with the host receptor could induce conformational changes that expose a trypsin cleavage site(s). the resulting cleavage might expose or activate the fusion peptide and activate pedv entry and pathogenesis. plaque formation by influenza viruses in the presence of trypsin characterization of the infectious salmon anemia virus fusion protein structural basis for paramyxovirus-mediated membrane fusion mutational analysis of the murine coronavirus spike protein: effect on 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of cellfusing activity of virions by trypsin and separation of two different k cleavage fragments evolution and ecology of influenza a viruses nucleotide sequence and expression of the spike (s) gene of canine coronavirus and comparison with the s proteins of feline and porcine coronaviruses the sars-cov s glycoprotein: expression and functional characterization porcine epidemic diarrhea virus (cv ) and feline infectious peritonitis virus (fipv) are antigenically related key: cord- -x z dg a authors: saito, kyoko; fukasawa, masayoshi; shirasago, yoshitaka; suzuki, ryosuke; osada, naoki; yamaji, toshiyuki; wakita, takaji; konishi, eiji; hanada, kentaro title: comparative characterization of flavivirus production in two cell lines: human hepatoma-derived huh . . - and african green monkey kidney-derived vero date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: x z dg a the flaviviridae is a family of enveloped viruses with a positive-sense single-stranded rna genome. it contains many viruses that threaten human health, such as japanese encephalitis virus (jev) and yellow fever virus (yfv) of the genus flavivirus as well as hepatitis c virus of the genus hepacivirus. cell culture systems highly permissive for the flaviviridae viruses are very useful for their isolation, propagation, and diagnosis, an understanding of their biology, and the development of vaccines and antiviral agents. previously, we isolated a human hepatoma huh- -derived cell clone, huh . . – , which is highly permissive to hepatitis c virus infection. here, we have characterized flavivirus infection in the huh . . – cell line by comparing with that in the african green monkey kidney-derived vero cell line, which is permissive for a wide spectrum of viruses. upon infection with jev, huh . . – cells produced a higher amount of virus particles early in infection and were more susceptible to virus-induced cell death than vero cells. similar outcomes were obtained when the cells were infected with another flavivirus, yfv ( d- strain). quantification of cellular and extracellular viral rna revealed that high jev production in huh . . – cells can be attributed to rapid viral replication kinetics and efficient virus release early in infection. in a plaque assay, huh . . – cells developed jev plaques more rapidly than vero cells. although this was not the case with yfv plaques, huh . . – cells developed higher numbers of yfv plaques than vero cells. sequence analysis of cdna encoding an antiviral rna helicase, rig-i, showed that huh . . – cells expressed not only a full-length rig-i mrna with a known dominant-negative missense mutation but also variants without the mutation. however, the latter mrnas lacked exon / − , indicating functional loss of rig-i in the cells. these characteristics of the huh . . – cell line are helpful for flavivirus detection, titration, and propagation. a a a a a the huh- (no. jcrb ) and vero (no. jcrb ) cell lines were purchased from the japanese collection of research bioresources (jcrb) cell bank, national institute of biomedical innovation (osaka, japan). huh . . - , huh . . , and huh- cells were maintained at ˚c in an atmosphere of % co in dulbecco's modified eagle's medium (dmem; wako pure chemical industries, osaka, japan; no. - ) supplemented with % (v/v) fetal bovine serum (fbs; sigma-aldrich, st. louis, mo, usa; no. - ml), . mm nonessential amino acids (nacalai tesque, kyoto, japan; no. - ), u/ml penicillin g, and μg/ ml streptomycin sulfate (nacalai tesque; no. - ). vero cells were maintained at ˚c in an atmosphere of % co in eagle's minimal essential medium (emem; wako pure chemical industries; no. - ) supplemented with % (v/v) heat-inactivated fbs, u/ml penicillin g, and μg/ml streptomycin sulfate. for comparative analysis, huh . . - and vero cells were grown in the same dmem-based medium as above but with heat-inactivated fbs. before starting the experiments, vero cells were adapted to this medium through at least eight passages. jev production in vero cells was comparable between the dmem-based and emem-based media (s a fig). the jev nakayama strain (isolated in japan in ) and jev rat strain [ ] were propagated in vero cells. the yfv d- strain, a current vaccine strain derived from the d strain [ ] , was kindly provided by dr. tomohiko takasaki (department of virology i, national institute of infectious diseases) and propagated in vero cells. the resultant culture supernatants were stored at − ˚c and used as virus stocks. these virus strains were chosen because they could be used in our biosafety level laboratory. for virus titration, a plaque assay was performed on vero cells according to the manual provided on the website of the national institute of infectious diseases [ ] with modifications. in brief, cell monolayers grown in -well plates (corning costar ; no. ) were inoculated with virus samples in . ml per well of culture medium with reduced heat-inactivated fbs ( %) and were incubated at ˚c for h. after removal of the samples, the cells were incubated in ml of overlay medium consisting of emem (nissui pharmaceuticals, tokyo, japan; no. ), . % (w/v) nahco , % (v/v) heat-inactivated fbs, mm l-glutamine (nacalai tesque; no. - ), and % (w/v) methylcellulose (tokyo chemical industry, tokyo, japan; no. m ) at ˚c for days. the cells were fixed with % (v/v) neutral buffered formalin (wako pure chemical industries; no. - ) and stained with . % (w/v) methylene blue. wells with − plaques were used to calculate the virus titer in plaque formation units (pfu) . for the comparative plaque assay, cells were similarly grown and infected, but overlaid with . ml per well of dmem (nissui pharmaceuticals; no. ) supplemented with . % (w/v) nahco , % (v/v) heat-inactivated fbs, mm l-alanyl-l-glutamine (nacalai tesque; no. - ), . mm nonessential amino acids, and . % (w/v) methylcellulose. jev plaque formation in vero cells was not so different between the dmem overlay medium and the emem overlay medium (s b fig). after fixation and staining, plates were scanned with a document scanner, and image data were obtained (s fig). to improve plaque visibility, an invert filter was applied to the image data, and highlights were adjusted using photoshop elements (version ; adobe systems, san jose, ca, usa). total rna was extracted and purified from cells and culture supernatant using the blood/cultured cell total rna mini kit (favorgen biotech, pingtung city, taiwan; no. fabrk - ) and the viral nucleic acid extraction kit i (favorgen biotech; no. favnk - ), respectively, according to the manufacturer's instructions. to determine the copy numbers of jev rna (nakayama strain; genbank accession no. ef . ), the rna fraction was subjected to one-step qrt-pcr using the thunder-bird probe one-step qrt-pcr kit (toyobo, osaka, japan; no. qrz- ) with primers # and and a hydrolysis probe # (table ). the following reactions were performed on the lightcycler system (roche applied science): reverse transcription at ˚c for min and denaturation at ˚c for min, followed by cycles of ˚c for s and ˚c for s. to normalize cellular jev rna levels, the copy numbers of mrna encoding glyceraldehyde- -phosphate dehydrogenase (gapdh) (human, genbank accession no. nm_ . ; chlorocebus sabaeus, genbank accession no. xm_ ) in the same rna samples were determined with primers # and and a hydrolysis probe # ( table ; the sequences of # − are homologous to both the human and monkey sequences) under the same qrt-pcr conditions, except that the annealing temperature was ˚c. a standard curve was established using serial dilutions of plasmids encoding the jev e protein (bases to ) or human gapdh. for construction of the plasmid encoding the jev e protein, the rna fraction prepared from a jev (nakayama strain) stock was reverse transcribed with primer # (table ) total rna from cells was reverse transcribed with an oligo dt primer using the primescript™ ii st strand cdna synthesis kit (described above). the first strand cdna was used as a template to amplify a cdna fragment encoding rig-i (ddx ) (genbank accession no. (table ) . upon sequence determination, nucleotide variations were excluded that were not consistent with the rna-seq data from huh . . (drr ) and huh . . - (drr ) [ ] or the whole-genome sequence data from huh- and huh . . - (ddbj dra database under the project id prjdb ) [ ] . all the experiments were done with three biological replicates and repeated as described in fig comparison of jev production between huh . . - , huh . first, the huh . . - cell line was compared with the parental huh . . and the ancestral huh- cell lines regarding jev production. cells were infected with jev (nakayama strain, unless otherwise noted), and then the amount of infectious virus particles (titer) in culture supernatant and jev-induced cell death were monitored during − d pi. the relative jev titer of these cell lines increased and peaked at − d pi in a similar manner ( fig a) . under the same seeding conditions, doubling times of huh . . - , huh . . , and huh- cells were not significantly different (after bonferroni correction, p > . ) (s b fig) . thus, jev production appeared to be comparable between the three cell lines and not enhanced in this lineage unlike for hcv production. in a parallel cell viability assay (fig b) , susceptibility to jevinduced cell death was not greatly different between the three cell lines. next, these cell lines were infected with jev, overlaid with methylcellulose-containing medium, and then compared regarding jev plaque formation. huh . . - and huh . . cells similarly developed jev plaques, whereas huh- cells began to die at d pi and detached from the surface of the well irrespective of infection at d pi ( fig c) . these results indicated that the huh . . - and huh . . cell lines are better than the huh- cell line for a jev plaque assay. although huh . . - and huh . . cell lines were comparable in terms of jev infection, the huh . . - cell line has the advantage of being phenotypically more stable than the huh . . cell line [ ] . thus, we hereafter focused on the huh . . - cell line and compared it with the vero cell line regarding jev production. jev production in huh . for comparative analysis, we used the vero cell subline jcrb , in which jev production was almost equivalent to or slightly higher than that in other vero cell sublines (s fig). huh . . - and vero cells under high and low confluency were infected with jev at moi . , and then the virus titer in culture supernatant was monitored from to d pi (fig ) . the culture supernatant of huh . . - cells exhibited a higher relative virus titer than that of vero cells, particularly during the early infection times. the difference in the relative virus titer was remarkable in high-cell-density infection (fig a) compared with low-cell-density infection ( fig b) , implying that cell-cell contacts promote viral spread in huh . . - cells. the difference in the titer was not accounted for by the growth rates of the two cell lines, because their doubling times under high confluency conditions were nearly equal (s fig). a parallel viability assay showed that huh . . - cells lost viability faster than vero cells irrespective of cell density (fig ) . the same trends were observed in jev production and cell viability at smaller moi ( . ), although high confluency appeared to be needed (s fig) . these results showed that jev production in the huh . . - cell line is higher than that in the vero cell line during early infection times and that the huh . . - cell line is more susceptible to jev-induced cell death than the vero cell line. the particle-to-pfu ratio was calculated for huh . . - -and vero-derived jev particles, and the ratio of the values of both particles was determined (fig ) . both virus particles showed a similar particle-to-pfu ratio, ruling out the possibility that huh . . - -derived virus particles had a higher infectivity than vero-derived ones. the reason for large variations found at and d pi remains unknown, but that at d pi may have been partly due to variable amounts of non-infectious viral rna leaking from dead huh . . - cells (fig ) . in a representative experiment (s fig) , the actual value of the particle-to-pfu ratio was minimally~ , which is roughly consistent with a previous report [ ] . taken together with the results of fig , these results suggested that huh . . - cells produce a higher amount of jev particles than vero cells during early infection times. next, we compared the jev replication kinetics between huh . . - and vero cells by monitoring intracellular and extracellular viral rna levels during − h pi. an initial rise in intracellular viral rna level in huh . . - cells was detected as early as h pi, whereas the rise for vero cells was not remarkable during - h pi (fig a [a]) . similarly, the level of extracellular viral rna from huh . . - cells rose earlier than that from vero cells (fig b [a] ). although the level of intracellular viral rna was not different between the two cell lines after h pi (fig a [b] ), the level of extracellular viral rna was higher for huh . . - cells than for vero cells during − h pi (fig b [b] ). furthermore, the relative amount of extracellular viral rna expressed as a percentage of the total (extracellular plus intracellular) amount of viral rna was about -fold higher in huh . . - cells than in vero cells at d pi (fig ) . thereafter, the value for huh . . - cells varied with each experiment, possibly due to variation in virus-induced cell death, but leading to no difference between the values for for both panels, bars with error bars represent the mean ± standard deviation (sd) of three independent experiments. statistical significance between cell lines was determined by a one-sample t test (for comparison with a hypothetical value, ) or an unpaired two-tailed t test using bonferroni correction and p values less than . were considered statistically significant. ns, not significant. (c) cells were seeded at x cells per well of a -well plate one day before infection and then infected with jev at pfu (determined with vero cells) per well. the cells were fixed and stained at − d pi. values given below the images are plaque numbers expressed as the mean ± sd of triplicates from one representative experiment. statistical significance was determined as described above. values in parentheses are p values, and those less than . were considered statistically significant. similar results were obtained in two other independent experiments (s fig). https://doi.org/ . /journal.pone. .g huh . . - and vero cells. thus, huh . . - cells may release jev more efficiently than vero cells at least at d pi. collectively, these results suggested that rapid viral replication kinetics and efficient early virus release contribute to high jev production in huh . . - cells. jev plaque formation (size and number) were compared between huh . . - and vero cell lines at − d pi. as shown in fig a and s a fig, clearly visible plaques were developed in huh . . - cells at as early as d pi, but not in vero cells at that time. in the course of the comparison of flavivirus production between huh . . - and vero cell lines infection, plaques in huh . . - cells grew more rapidly than those in vero cells. similar results were observed when the cells were infected with another jev strain, rat (fig b and s b fig) . these results showed that the huh . . - cell line supports more rapid jev plaque formation than the vero cell line. the rapid plaque formation in huh . . - cells appears to reflect their high jev productivity and susceptibility to jev-induced cell death. the plaque numbers of the nakayama strain at d pi were comparable between the two cell lines (fig a and s a fig), whereas those of the rat strain tended to be higher in the huh . . - cell line than in the vero cell line (fig b and s b fig) . these results indicated that the difference in plaque numbers between the two cell lines varies by each virus strain. fig , and the culture supernatants were harvested at the indicated times. rna was extracted and purified from each culture supernatant, and viral rna copy number in the supernatant was determined by qrt-pcr and regarded as the number of virus particles. a virus titer in pfu of each culture supernatant was also determined by plaque assay, and particle-to-pfu ratio was calculated. each symbol represents the relative value calculated by dividing the value of huh . . - -derived jev by that of vero-derived jev obtained from the same experiment. bars with error bars represent the mean ± sd of the ratio from eight ( and d pi) or seven ( d pi) independent experiments (the reason for the different sample sizes was that one experiment lacked data for d pi). statistical significance was determined by a one-sample t test. values in parentheses indicate p values, and those less than . were considered statistically significant. (fig c and d) . the combined data showed that virus yield in huh . . - cells was significantly higher than that in vero cells for high confluency at d pi and low confluency at and d pi. furthermore, huh . . - cells were more susceptible to yfv-induced cell death than vero cells (fig e and f ), as observed with jev. these results suggested that the huh . . - cell line, compared with the vero cell line, has a higher virus productivity and susceptibility to virus-induced cell death upon flavivirus infection. next, we compared the yfv plaque formation between the two cell lines (fig and s fig) . yfv plaques began to appear at d pi in both cell lines. although plaques in huh . . - cells were slightly smaller than those in vero cells, huh . . - cells developed a several fold higher number of yfv plaques than vero cells. these results suggested that the high yfv productivity and susceptibility to yfv-induced cell death of huh . . - cells can be mainly attributed to their high infection efficiency. although yfv plaques developed in vero cells at d pi in fig , no cell death was observed with these cells at this time point in fig e and f . a possible explanation for this discrepancy in cell death may be the difference in culture conditions: the serum concentration was % for fig and % for fig . in addition, the medium in the case of jev, the lower the serum concentration is, the more cell death occurs [ ] . recently, codon in the rig-i gene from the huh . . - cell line was found to be heterozygous: mutant-type ata (ile) and wild-type aca (thr) [ ] . to confirm whether the huh . . - cell line is defective in rig-i, we amplified the rig-i cdna derived from total rna isolated from huh- , huh . . and huh . . - cells. an amplicon corresponding to the expected size for the rig-i sequence (~ kbp) and another, with a smaller size (~ kbp), were detected in the three cell lines (fig a) and directly sequenced (fig b) . the -kbp and -kbp cdnas from the huh- cell line exclusively contained the wild-type codon . in contrast, the -kbp cdnas from the huh . . and huh . . - cell lines contained the mutanttype codon , whereas the -kbp cdnas from these cell lines contained the wild-type codon . cloning and sequencing of the cdnas of huh- and huh . . - cell lines revealed that the -kbp cdnas were full-length (fl , ), whereas the -kbp cdnas lacked exons − (sv found in both cell lines) or − (sv found in huh . . - ) (fig c and s fig) . rig-i protein isoforms deduced from the splice variants (sv , ) completely lacked a helicase atpbinding domain, indicating that they are defective like rig-i protein with a mutated atpbinding site [ ] . these results were consistent with the above findings [ ] and indicated that huh . . - is a rig-i null mutant cell line. although the huh . . - cell line is more permissive to hcv infection than the huh . . cell line [ ] , we found that this was not the case with jev infection: the huh . . - and the parental huh . . cell lines were comparable in terms of jev productivity, susceptibility to jev-induced cell death, and jev plaque formation (fig ) . even the ancestral huh- cell line was comparable except for plaque formation. these findings indicate that genetic factors contributing to the high permissiveness of the huh . . - cell line to hcv infection are specific rather than broadly proviral. one such genetic factor might be involved in stable hcv receptor expression [ ] . unlike the huh . . - and huh . . cell lines, the huh- cell line was not maintained under our culture conditions for the plaque assay (fig c) , likely because our huh- cells had a lower fitness to cell culture than huh . . - and huh . . cells. here, we found three differences in outputs of flavivirus infection between huh . . - and vero cell lines. first, huh . . - cells produced higher amounts of infectious jev and yfv than vero cells early in infection (figs and a- d ). in the case of jev, the high virus productivity of huh . . - cells may be attributed not to the increase in infectivity of a virus particle (fig ) , but to viral rapid replication kinetics and efficient virus release early in infection (figs and ) . therefore, huh . . - cells are more suitable than vero cells for obtaining large amounts of flavivirus in a short period of time. recently, huh- cells were shown to produce higher amounts of zika virus than other cells including vero e cells (atcc crl- ) early in infection [ ] . considering that jev production in huh . . - cells and the ancestral huh- cells were comparable (fig a) , high virus productivity early in flavivirus infection may be a common feature among the huh- lineage. in addition, a similar trend was observed when huh- cells were infected with middle east respiratory syndrome coronavirus [ ] . thus, the feature may not be limited to flavivirus infection. second, we found that huh . . - cells were more susceptible to jev/yfv-induced cell death than vero cells (figs , e and f ). these characteristics of the huh . . - cell line may facilitate cell viability-based screenings of antiviral host factors and agents for flaviviruses. meanwhile, the potent cell death observed in huh . . - cells appears to be a part of the reason for plateaued or reduced virus production late in infection. therefore, genetic engineering of the huh . . - cell line to make it less susceptible to virus-induced cell death might yield a cell line with more flavivirus production. fig . (a, b) culture supernatants were harvested at , , , and h pi, and virus titers in the supernatants were determined by plaque assay. each point represents the mean ± sd of triplicates from one representative experiment. some error bars are not visible due to their small size. similar results were obtained in two other independent experiments (s fig). (c, d) the titer ratio was calculated by dividing the titer value of huh . . - cells by that of vero cells at each time point. bars with error bars represent the mean ± sd of three independent experiments (a, b and s fig). each symbol represents the mean from one experiment. (e, f) cell viability was determined at the indicated times. bars with error bars represent the mean ± sd of three independent experiments. statistical significance was determined by an unpaired two-tailed t test (a, b, e, f) or a one-sample t test (c, d). values in parentheses indicate p values, and those less than . were considered statistically significant. huh, huh . . - cells. https://doi.org/ . /journal.pone. .g comparison of flavivirus production between huh . . - and vero cell lines third, we found that huh . . - cells developed jev plaques rapidly (fig ) and formed a larger number of yfv plaques (fig ) compared with vero cells, though the difference in plaque numbers between the two cell lines varied by each virus. the jev plaque phenotype of huh . . - cells appears to reflect their high virus productivity and susceptibility to virusinduced cell death. because a plaque assay is somewhat time-consuming, the plaque phenotype observed with jev infection in huh . . - cells can be utilized to shorten the period of plaque assay. in addition, the high-number plaque phenotype observed with yfv infection in huh . . - cells can be utilized to improve the sensitivity of detection and titration of yfv by comparison of flavivirus production between huh . . - and vero cell lines plaque assay. vero cells may be more suitable to viruses requiring long-term cultivation to grow plaques, because they were more tolerant to our plaque assay conditions than huh . . - cells. in addition, the higher tolerance of vero cells to flavivirus-induced cell death, compared with huh . . - cells, may be a feature that makes the vero cell line advantageous for vaccine production. on top of these findings, we provided evidence that huh . . - is a rig-i null mutant cell line. our results suggested that, in a huh . . - cell, one allele of the rig-i gene generates full-length mrna with a t i mutation that abolishes rig-i-mediated antiviral signaling, whereas the other allele generates defective splice variants without the mutation. one of the splice variants is also expressed in the parental huh- cell line. codon of rig-i gene of huh . . cells was heterozygous as was that of huh . . - cells, showing that the heterozygosity of the codon was stable at least during the time span for isolation of the huh . . - clone from huh . . cells. at present, the underlying mechanisms that generate splice variants and their impacts on rig-i signaling remain to be studied. although jev has been restricted by rig-i in a mouse model [ ] and mouse cell lines [ , ] , jev production and susceptibility to jev-induced cell death were not dramatically altered in the huh . . - and huh . . cell lines compared with the huh- cell line (fig a and b) , implying that the rig-i pathway may not work or may be counteracted by viral mechanisms [ ] under our experimental conditions. in conclusion, our study highlighted the characteristics of the huh . . - cell line that are helpful for improvement of flavivirus propagation, detection, and titration. further study is needed to investigate the potential versatility of the huh . the culture supernatants were harvested at the indicated times. rna was extracted and purified from each culture supernatant, and viral rna copy number in the supernatant was determined by qrt-pcr. a virus titer in pfu of each culture supernatant was also determined by plaque assay, and particle-to-pfu ratio was calculated. each point represents the mean ± sd of triplicates from one representative experiment. statistical significance was determined by an unpaired two-tailed t test. values in parentheses indicate p values, and those less than . were considered statistically significant. huh, huh . (sv , ) . alignment of nucleotide sequences was performed using genetyx version . . (genetyx co., tokyo, japan). (pdf) s fig. unprocessed image data, related to figs c, , , s b, s , s and s . (pdf) s raw images. raw image data (fig a) . (pdf) estimated global incidence of japanese encephalitis: a systematic review fact sheets yellow fever studies on sv in tissue culture: preliminary step for cancer reserach in vitro studies on sv in tissue culture: preliminary step for cancer research in vitro biological characteristics and viral susceptibility of an african green monkey kidney cell line (vero) the genome landscape of the african green monkey kidney-derived vero cell line homozygous deletion of the alpha-and beta -interferon genes in human leukemia and derived cell lines guidelines for plaque-reduction neutralization testing of human antibodies to dengue viruses west nile virus infection and serologic response among persons previously vaccinated against yellow fever and japanese encephalitis viruses. vector borne zoonotic dis cell substrates for the production of viral vaccines the vero cell-derived, inactivated, sa - - strain-based vaccine (ixiaro) for prevention of japanese encephalitis growth of human hepatoma cells lines with differentiated functions in chemically defined medium highly permissive cell lines for subgenomic and genomic hepatitis c virus rna replication robust hepatitis c virus infection in vitro production of infectious hepatitis c virus in tissue culture from a cloned viral genome regulating intracellular antiviral defense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i roles of rig-i n-terminal tandem card and splice variant in trim -mediated antiviral signal transduction viral rna detection by rig-i-like receptors rig-i in rna virus recognition isolation and characterization of an huh. . . -derived cell clone highly permissive to hepatitis c virus characterization of the e- (glu/lys) mutation in japanese encephalitis virus by using a stable, full-length, infectious cdna clone the effect of prolonged cultivation in vitro upon the pathogenicity of yellow fever virus efficient trafficking of ceramide from the endoplasmic reticulum to the golgi apparatus requires a vamp-associated protein-interacting ffat motif of cert identification of characteristic genomic markers in human hepatoma huh and huh . . - cell lines development of multiplex real-time reverse transcriptase pcr assays for detecting eight medically important flaviviruses in mosquitoes flavivirus activates phosphatidylinositol -kinase signaling to block caspasedependent apoptotic cell death at the early stage of virus infection regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp comparative analysis of different cell systems for zika virus (zikv) propagation and evaluation of anti-zikv compounds in vitro mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment differential roles of mda and rig-i helicases in the recognition of rna viruses rig-i mediates innate immune response in mouse neurons following japanese encephalitis virus infection roles of tlr and rig-i in mediating the inflammatory response in mouse microglia following japanese encephalitis virus infection japanese encephalitis virus ns inhibits type i interferon (ifn) production by blocking the nuclear translocation of ifn regulatory factor and nf-kap-pab we thank dr. francis v. chisari (the scripps research institute, la jolla, ca, usa) for providing the huh . . cell line. we also thank dr. tomohiko takasaki (department of virology i, national institute of infectious diseases) for providing the yfv d- strain. key: cord- -n knqj z authors: su, yunfang; hou, yixuan; wang, qiuhong title: the enhanced replication of an s-intact pedv during coinfection with an s ntd-del pedv in piglets date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: n knqj z porcine epidemic diarrhea virus (pedv) variants having a large deletion in the n-terminal domain of the s subunit of spike (s) protein were designated as s ntd-del pedvs. they replicate well in experimentally infected pigs. however, on farms they often co-infect pigs with the pedv containing an intact s protein (s-intact pedv). we aimed to characterize viral replication and pathogenesis in neonatal gnotobiotic pigs infected simultaneously with the two types of pedv using two recombinant pedvs: icpc a and its s ntd-del form icpc a-s Δ . additionally, viral replication was compared in vero and ipec-dq cells at the presence of bovine mucin (bm), porcine gastric mucin (pgm), swine bile and bile acids during inoculation. in the pigs coinfected with icpc a and icpc a-s Δ , icpc a replicated to a higher peak titer than its infection of pigs without the presence of icpc a-s Δ . the severity of diarrhea and intestinal atrophy were similar between icpc a and the coinfection groups, but were significantly higher than icpc a-s Δ group. in vero and ipec-dq cells, certain concentrations of bm, pgm, bile and bile acids increased significantly the infectivity of icpc a but had no or negative effects on icpc a-s Δ . these results indicated that the replication of the s-intact pedv was enhanced during coinfection in piglets. this observation may be explained partially by the fact that mucin, bile and bile acids in gastrointestinal tract had facilitating effects on the infection of s-intact pedv, but no/inhibition effects on s ntd-del pedv. porcine epidemic diarrhea virus (pedv) belongs to the genus alphacoronavirus in the family coronaviridae. it causes severe gastroenteritis in neonatal pigs worldwide. the genome size of pedv is kb. it contains orf a and orf b encoding - non-structural proteins, an orf encoding an accessory protein, and four orfs encoding structural proteins: spike (s), membrane (m), envelop (e), and nucleocapsid (n) proteins. pedv s protein mediates the essential functions of receptor binding (via s subunit) and subsequent fusion of the viral and cellular membranes (via s subunit) (li, ) . s subunit contains two domains, the n-terminal domain (s ntd, residues - based on pedv cv ) and the c-terminal domain (s ctd, residues - ). however, the cellular receptor of pedv is still unknown (li et al., b; shirato et al., ) . although the sialic acid binding activity of s -ntd was observed in many coronaviuses and facilitated virus replication (schwegmann-wessels et al., ; li et al., ; desmarets et al., ) , s -ntd is dispensable for some mutants of transmissible gastroenteritis coronavirus (tgev) and pedv (schwegmann-wessels et al., ; oka et al., ; suzuki et al., ; hou et al., ) . pedv variants with a big deletion in the s -ntd have been designated as s ntd-del pedvs (hou et al., ) . they have been detected in field pig samples, indicating sufficient replication of these s ntd-del pedvs in vivo (suzuki et al., ; diep et al., ; zhang et al., a; su et al., ) . interestingly, most field s ntd-del pedvs exist as a member of coinfection of pigs with the s-intact pedv (diep et al., ; su et al., ) . however, the mechanisms and consequences of the coinfection have not been investigated. studies showed that under unfavorable conditions as encountered in the intestinal tract, sialic acid-binding of tgev played an important role in viral replication. when absorption time was reduced, tgev infectivity was also reduced in the cells whose sialic acids were removed (schwegmann-wessels et al., ) . consequently, sialic acid binding activity may facilitate the infection of tgev by helping the virus resist detergent-like substances encountered during passing through the gastrointestinal tract (krempl et al., ) . therefore, we hypothesized that mucin, rich in sialic acid, may enhance the infection of pedv. interestingly, bile acids can induce or promote the replication of some enteric viruses like a porcine sapovirus (chang et al., ) and human noroviruses (ettayebi et al., ) . since intestinal epithelial cells are exposed to bile acids, we hypothesized that bile acids promote pedv infection. previously, we generated two recombinant pedvs, pedv icpc a and icpc a-s Δ (hou et al., ) . pedv icpc a is an infectious cdna clone-derived virus of the emerging highly virulent pedv pc a strain. on the other hand, pedv icpc a-s Δ is a mutant version of icpc a bearing a -aa deletion (residues - ) in the s ntd region, corresponding to the s domain (s °) of the spike protein (li et al., a; hou et al., ) . in this study, we investigated the replication and pathogenesis of the two viruses during coinfection in neonatal gnotobiotic (gn) piglets. some intestinal substances (mucin, bile and bile acids) were tested for their effects on the infection of the individual viruses in vitro. we selected two cell lines, vero and ipec-dq cells. vero cells are the most commonly used cell line for pedv isolation and propagation (teeravechyan et al., ) . ipec-dq cell is a subclone of porcine small intestinal cell line ipec-j (zhang et al., b) . although pedv grows less efficiently in ipec-dq cells than in vero cells, pedv infection of ipec-dq cells is more relevant physiologically to pedv natural infection in the intestines of pigs. vero cells (atcc number ccl ) were cultured in dulbecco's modified eagle's medium (dmem, gibco, carlsbad, ca, usa) supplemented with % fetal bovine serum (fbs, hyclone, logan, ut, usa) and % penicillin/streptomycin (gibco). for virus propagation in vero cells, the maintenance medium was dmem supplemented with . % tryptose phosphate broth solution (tpb, sigma, st. louis, mo, usa), % penicillin/streptomycin (gibco) and μg/ml trypsin ( . % trypsin, gibco). ipec-dq cell line was a gift from dr. dongwan yoo, university of illinois at urbana-champaign, urbana il, usa. it was a subclone of ipec-j cells, a porcine small intestinal cell line, and maintained in roswell park memorial institute medium (rpmi- , gibco) supplemented with % fbs and % penicillin/streptomycin (zhang et al., b) . for virus propagation in ipec-dq cells, maintenance medium was rpmi- containing . % tpb, % penicillin/streptomycin and μg/ml trypsin. two recombinant pedv, icpc a and icpc a-s Δ , were generated previously by our lab using the infectious clone of pedv pc a strain (hou et al., ) . neuraminidase (na, from clostridium welchii), porcine gastric mucin (pgm) and several bile acids [(glycochenodeoxycholic acid (gcdca), chenodeoxycholic acid (cdca), deoxycholic acid (dca), and ursodeoxycholic acid (udca)] were all purchased from sigma. bovine mucin (bm) was purchased from worthington (worthington biochemical corp., lakewood, nj, usa). swine bile was collected from a pregnant sow after c-section surgery. the mouse monoclonal antibodies (mab) h , f and a targeting the s °domain of pedv s protein (fig. s a ) was provided by dr. berend jan bosch, utrecht university, utrecht, netherlands (li et al., a) . the mab sd - targeting pedv nucleocapsid (n) protein was provided by drs. eric nelson and steven lawson, south dakota state university. the guinea pig polyclonal antibody (pab) gp targeting the s subunit of the s protein of pedv pc strain (s Δ ) (fig. s a) , rabbit pab rb targeting pedv n protein and swine pedv-positive serum (virus neutralization titer : ) were generated by our laboratory previously (hou et al., ) . . . duplex rt-qpcr method for the detection and differentiation of pedv icpc a and icpc a-s Δ based on the nucleotide sequence differences in the s ntd coding region between the icpc a and icpc a-s Δ , a duplex taqman real-time reverse-transcription quantitative pcr (rt-qpcr) was developed. we designed the primers and probes (table , fig. s b ) and had them synthesized by integrated dna technologies (skokie, il, usa). the rna of the two viruses was extracted using rneasy mini kit (qiagen, valencia, ca, usa). to generate a standard dna, a kit of superscript™ iii first-strand synthesis supermix (thermo fisher scientific, carlsbad, ca, usa) was used for cdna synthesis following manufacturer's instructions. primer set pedv- f and pedv- r (oka et al., ) was used to amplify a . kb or a . kb fragment covering pedv partial nsp gene and s gene of icpc a and icpc a-s Δ , respectively. the pcr products were purified using gel extraction kit (qiagen) and used as standard dna. the reaction consisted with μl × pcr buffer, . μl mm dntps (qiagen), . μl each primer ( μm), . μl each probe ( μm), . μl rnasin (promega, wi, usa), . μl enzyme mix (qiagen), . μl each dna/rna and . μl sterilized water. the rt-qpcr procedure was: °c for min, °c for min and cycles of °c for s and °c for s. we generated two standard curves using light-cycler® system (roche life science, indianapolis, in, usa). the two standard curves for the serially diluted dna fragments of icpc a and icpc a-s Δ showed regressions with coefficients of - . and - . , respectively, and the corresponding correlation coefficient (r ) of . and . , respectively. the primers and probes were further tested using individual and the mixture of two viruses. we tested the correlation between s gene titers and infectious titers of pedv icpc a and icpc a-s Δ using serially diluted, cell cultured viruses (table s and s ). for the samples of mixed viruses, the duplex rt-qpcr was performed to differentiate the s gene of each virus. the detection limit was log s gene copies/ml for both icpc a and icpc a-s Δ . the gn piglets were derived and raised as described previously (saif et al., ) . thirty-one gn piglets were assigned to five experimental groups: ( ) icpc a (n = ; pfu/pig); ( ) icpc a-s Δ (n = ; pfu/pig); ( ) coinfection of icpc a and icpc a-s Δ (n = ; pfu/pig of each virus); ( ) mock (n = ; pbs); ( ) second passage of coinfection [n = ; small intestinal contents (sic) (sample #pe ) of one piglet in group collected at day post inoculation (dpi), at a dose of log copies/ml based on the n gene, log copies/ml and log copies/ml based on the icpc a-specific s gene and icpc a-s Δ -specific s gene, respectively, corresponding to table primers and probes of the duplex rt-qpcr. log pfu/ml of infectious pedv] . at days of age, piglets in groups - were orally inoculated with individual or the mixture of the viruses. the group pig was inoculated at days of age and was euthanized at dpi. to examine histopathological changes, one or two piglets of groups - were euthanized at . dpi and - dpi, respectively. after inoculation, all piglets were evaluated daily for clinical signs, such as diarrhea, vomiting, anorexia, and depression. fecal consistency was examined by collecting rectal swabs and scored as follows: , solid; , pasty; , semiliquid (mild diarrhea); and , liquid (severe diarrhea). total fecal rna was extracted using magmax- rna isolation kit (thermo fisher scientific), and the viral rna was titrated by the rt-qpcr targeting the n gene and the duplex rt-qpcr targeting the s gene of each virus. fecal infectious pedv shedding titers were tested using vero cells in -well plates and determined as % tissue culture infective doses (tcid ) (reed and muench, ) . at necropsy, three sections of the jejunum (proximal, middle, and distal) and one section of the ileum were collected and fixed in . % formalin (fisher scientific co llc, florence, ky, usa). all tissues were trimmed, processed, embedded in paraffin, and sectioned using routine procedures . to compare the pedv-specific histopathological changes among different groups of pigs, ihc staining was performed as described previously for the detection of pedv n proteins using mab sd - as the primary antibody (hou et al., ) . the ihc staining was carried out using a nonbiotin polymerized horseradish peroxidase system (biogenex laboratories, san ramon, ca, usa). finally, tissues were counterstained with hematoxylin. images of tissues were observed and captured by olympus ix- fluorescent microscope (center valley, pa, usa). ratios of villous height to crypt depth (vh/ cd) of the jejunum and ileum of individual piglets were measured using a computerized imaging system (metamorph, olympus, japan) as described previously . for each intestinal section, villi and crypts were measured. to detect and differentiate icpc a-and icpc a-s Δ -infected cells in the coinfected pigs, if staining was performed. the slide preparation steps were as same as those of the above ihc procedure. the mixture of mouse mabs h , f and a ( : in pbs) (li et al., a) targeting the s °domain of pedv s protein was used as the primary antibody for the staining of icpc a-infected cells only. tissues were incubated at ℃ overnight, after three washings with pbst (pbs with . % tween ), alexa fluor -conjugated goat antimouse igg (h + l) (thermo fisher scientific) ( : diluted in pbs) was used as the second antibody and tissues were incubated at room temperature for h. after washing with pbst, guinea pig pab gp ( : diluted in pbs), targeting the s Δ and was for the staining of both viruses, was added. then, the slides were incubated at room temperature for h. after washing three times with pbst, alexa fluor -conjugated goat anti-guinea pig igg (h + l) (thermo fisher scientific) ( : diluted in pbs) was used as the nd antibody and the slides were incubated at room temperature for h. after washing with pbst, mg/ml dapi ( ′, -diamidino- -phenylindole, dihydrochloride, roche life science, indianapolis, in, usa) was added for nucleic acid staining. finally, an autofluorescence quenching kit (vector® trueview™, burlingame, ca, usa) was used to quench the autofluorescence of the tissues. images of stained tissues were captured by olympus ix- fluorescent microscope. red, green and blue fluorescence staining was merged by imagej software (https://imagej.nih. gov). . . isolation of the pig-passaged icpc a and icpc a-s Δ from the sic of a pig coinfected with the viruses the sic (sample #pe ) of a coinfected piglet in group was positive for both viruses by the duplex rt-qpcr. virus isolation was performed using plague assays (oka et al., ) . the sic was diluted in dmem and the % suspension was vortexed briefly followed by centrifugation at , ×g for min at ℃. a series of -fold dilutions ( − - − ) of the supernatants were prepared in dmem (containing . μg/ml trypsin) and were used immediately for inoculation of vero cell monolayers in -well plates ( μl per well). after incubating at ℃ for h, the inoculum was removed and the monolayers were washed once with pbs. then ml/well of agarose overlay was added. the plate was kept for days before picking up clones. for propagating each clone, vero cell monolayers in -well plates were prepared and individual plagues were picked and added into individual wells. at dpi, rna was extracted and the duplex rt-qpcr method specific for both viruses was performed. . . multi-step growth kinetics and if staining of coinfection of pedv icpc a and icpc a-s Δ in vero and ipec-dq cells the growth kinetics of icpc a-s Δ and icpc a during the coinfection of the two viruses were investigated in vero and ipec-dq cells. cell monolayers on -well plates were inoculated with one of the three inocula: ( ) . multiplicity of infection (moi) of icpc a; ( ) . moi of icpc a-s Δ ; and ( ) . moi of icpc a and . moi of icpc a-s Δ . viruses were diluted in dmem containing μg/ml trypsin and were added to cell monolayers. after incubating at ℃ for h, the inoculum was removed and the cell monolayers were washed for three times. then the plates were added with the maintenance medium and incubated at ℃. the samples were collected at different time points ( h, h, h, h, h) and frozen (- ℃) and thawed once before testing. rna was extracted using the magmax- rna isolation kit (thermo fisher scientific) and virus titers were tested by the duplex rt-qpcr specific for both viruses. for if staining of s proteins at h post infection (hpi), the procedure in . section was performed. . . infection of pedv icpc a and icpc a-s Δ in na-treated vero and ipec-dq cells vero/ipec-dq cell monolayers in -well plates were treated with mu na in dmem/rpmi- or mock dmem/rpmi- at ℃ for h. after three washings with dmem/rpmi- , cells were inoculated with icpc a or icpc a-s Δ at a moi of . with μg/ml trypsin for h. next, the inoculum was removed followed by three washings. dmem/rpmi- containing μg/ml soybean trypsin inhibitor (sbti) and swine pedv positive serum (virus neutralization titer : , : dilution) was added to prevent virus spreading. cells were fixed with acetone-methanol ( : ) at hpi. the numbers of pedv-infected cell foci were determined by if assays as described for the staining of pedv n proteins (hou et al., ) . briefly, rabbit pab rb ( : ) was used as the primary antibody. after incubating at ℃ overnight, cells were washed three times. then alexa fluor -conjugated goat anti-rabbit igg (h + l) (thermo fisher scientific) ( : ) was used as the nd antibody and the cells were incubated at room temperature for h. after washing and adding mounting medium, cells were observed and picture were captured by using olympus ix- fluorescent microscope. the numbers of fluorescent focus units (ffu) were counted by imagej software. the percentages of ffu were calculated by the value of mock group as %. . . effect of mucin, bile and bile acids on the infection of pedv icpc a and icpc a-s Δ in vero and ipec-dq cells viruses (icpc a or icpc a-s Δ ) were mixed with different concentrations of bm ( , . , . , . mg/ml) or pgm ( , . , . , . , . mg/ml). then μg/ml trypsin were added to the mixture. monolayer of vero/ipec-dq cells in -well plates were inoculated with each virus mixture at a moi of . . the plates were incubated at ℃ for h, then the inoculum was removed. after three washings, dmem/rpmi- containing μg/ml sbti and swine pedv positive serum ( : dilution) was added. cells were fixed at hpi. the numbers of ffu were determined by if assay for the staining of pedv n proteins (see section . ). the percentages of ffu were calculated by the value of mock group as %. similarly, different concentration of swine bile ( , . , . , . , . %) and bile acids (gcdca, cdca, dca, and udca) at , , , , μm were tested. statistical analysis was performed using graphpad prism software (graphpad software, la jolla, ca, usa). the experimental data were analyzed by one-way anova and student`s t-test. data are shown as m ± sd, and a p < . was considered as significant. . . the replication of icpc a was eventually enhanced during coinfection with icpc a-s Δ in neonatal gn piglets as shown in fig. , all pedv-inoculated pigs but no pigs in the mock group had diarrhea and shed pedv. the fecal consistency scores and peak fecal pedv n and s rna shedding titers of piglets in the coinfection group reached the peak at . dpi, which was delayed half a day compared with the icpc a group ( dpi) but earlier than the icpc a-s Δ group (> dpi). by dpi, all pigs in the icpc a and coinfection groups but no pigs in the icpc a-s Δ group died or showed moribund. compared with the peak fecal pedv n gene shedding titer ( . ± . log copies/ml) of piglets in the icpc a group ( dpi), pigs in the coinfection group had a significantly higher peak titer ( . ± . log copies/ml) ( fig. b and table ) at a delayed time point ( . dpi). in addition, the peak fecal infectious pedv shedding titer of the coinfection group ( . ± . log tcid /ml) were significantly higher than that of the single infection groups ( . ± . and . ± . log tcid /ml of icpc a and icpc a-s Δ groups, respectively) ( table ) . interestingly, the peak s gene shedding titers of icpc a ( . ± . log copies/ml) in the coinfection group at . dpi was significantly higher than that of icpc a group ( . ± . log copies/ml) at dpi (fig. c, table ). however, the peak s gene shedding titers ( . ± . log copies/ml) of icpc a-s Δ in the coinfection group was significantly lower than that of the icpc a-s Δ group ( . ± . log copies/ml) (fig. c , table ). in the second passage of coinfection, only icpc a but not icpc a-s Δ s gene was detected in the large intestinal contents (lic) of the piglet, which was orally inoculated with the sic (sample #pe ) of one pig in the coinfection group and was positive for both viruses. the lic had a virus titer of . log tcid /ml for infectious pedv, . log s gene copies/ml and . log n gene copies/ml for icpc a. histopathological examination was performed and villous atrophy was observed in the jejunum and ileum of piglets in all pedv-inoculated groups ( fig. a and b ). the small intestinal villi in the icpc a-s Δ group had a milder villous atrophy than those in the icpc a and coinfection groups. the vh/cd ratios of jejunum and ileum of all the pedv-inoculated groups were significantly lower than those of the mock group (fig. b) . the ratios of the icpc a and coinfection groups were similar and were significantly lower than that of the icpc a-s Δ group. ihc staining showed that no pedv-positive intestinal epithelial cells were observed in all pedv-inoculated pigs fig. . evaluation of the replication of icpc a and icpc a-s Δ in gn pigs infected with individual or both viruses. (a) fecal consistency scores of individual gn piglets and the mean of each group were shown. the scores were named as follows: , solid (normal); , pasty (normal); , semiliquid (mild diarrhea); and , liquid (severe diarrhea). two piglets from icpc a, icpc a-s Δ and coinfection groups, and one pig from mock group were euthanized at hpi and - dpi, respectively, for histopathological examination. (b) fecal pedv n gene rna shedding titers (for both icpc a and icpc a-s Δ ) of each group. data are shown as mean (m) ± standard deviations (sd) of pigs in each group. (c) fecal pedv s gene rna shedding titles of each virus. data are shown as m ± sd. at . dpi (data not shown). at - dpi, a few pedv n antigens were observed in the small intestine of the icpc a-s Δ -infected piglets, while extensive antigens were stained in those of the icpc a and coinfection piglets ( fig. a) . by if staining for the pedv s proteins, a few pedv antigens were observed in the jejunum of the icpc a-s Δ -inoculated piglets (green alone, positive for s Δ only), while many antigens were observed in those of the icpc a-inoculated piglets [yellow, representing positive for both s Δ (green) and s °d omain (red)] and the coinfection piglets (yellow) (fig. c) . . . icpc a alone, but not icpc a-s Δ alone was isolated from the sic of one of the pigs infected with the two viruses simultaneously the sic of the one piglet in the coinfection group, which was positive for both viruses by the duplex rt-qpcr were used for virus isolation in vero cells using plague assays. we picked up plagues and found that plagues were positive for icpc a and plagues were positive for both viruses by the duplex rt-qpcr. no plagues were positive for s ntd-del pedv alone. during coinfection in vero cells, icpc a replicated to a titer of . ± . log copies/ml at hpi, which was similar to that of the single infection ( . ± . log copies/ml) (fig. a) . however, icpc a-s Δ s gene peak titer was . ± . log copies/ ml, which was significantly lower than that of the single infection ( . ± . log copies/ml). in vero cells, icpc a alone and icpc a-s Δ alone replicated to similar titers. similarly, during coinfection in ipec-dq cells, icpc a replicated to a similar titer to its single infection ( . ± . vs. . ± . log copies/ml at hpi (fig. c ). icpc a-s Δ s gene peak titer was . ± . log copies/ml, which was significantly lower than that of the single infection ( . ± . log copies/ml). in contrast to those results in vero cells, icpc a alone replicated to significantly higher titers than icpc a-s Δ alone in ipec-dq cells. by if staining, more icpc a-s Δ -infected cells (green) were observed in vero cells than in iepc-dq cells in both icpc a-s Δ alone and co-infection conditions ( fig. b and d) . at the second passage of the coinfection, icpc a rna and antigens were detected in both cells; however, icpc a-s Δ rna and antigens were detected exclusively in vero cells but not in ipec-dq cells (data not shown). . . the percentage of infectivity of icpc a was significantly lower than that of icpc a-s Δ in na-treated vero and ipec-dq cells after removing sialic acids from the cell surface using mu na in vero and ipec-dq cells, the percentages of infectivity of icpc a and icpc a-s Δ were reduced significantly (fig. ) . however, in both cells treated with na, the percentages of infectivity of icpc a were significantly lower than those of icpc a-s Δ . . . mucin enhanced the infectivity of icpc a but had no or inhibition effects on icpc a-s Δ in both vero and ipec-dq cells in vero cells, . - . , . and . mg/ml bm increased, had no effects and decreased the infectivity of icpc a, respectively (fig. a ). . - . mg/ml pgm increased the infectivity of icpc a . - . fold (fig. b) . however, the infectivity of icpc a-s Δ was unaffected or inhibited significantly by bm and pgm at the tested concentration ( fig. a and b) . in ipec-dq cells, the infectivity of icpc a was increased . - . fold and . - . fold by bm ( . - . mg/ml) and pgm ( . - . mg/ ml), respectively. however, there was no effects at the highest pgm concentration tested ( . mg/ml). on the other hand, low to medium concentrations of bm ( . - . mg/ml) and pgm ( . - . mg/ml) had no effects on the infectivity of icpc a-s Δ . only high concentrations of bm ( . mg/ml) and pgm ( . - . mg/ml) reduced the infectivity of icpc a-s Δ significantly ( fig. c and d ). . . bile and bile acids enhanced the infectivity of icpc a but had no or inhibition effects on icpc a-s Δ in vero cells, low to medium concentrations of swine bile ( . - . %) increased . - . fold of the infectivity of icpc a (fig. a) . (fig. b-e) . however, the infectivity of icpc a-s Δ was unaffected ( . - . %) or inhibited significantly by bile ( . - . %) by bile (fig. a) . bile acids a piglets in group were inoculated with pfu/pig of icpc a; piglets in group were inoculated with pfu/pig of icpc a-s Δ ; piglets in group were inoculated with pfu/pig of each virus; piglets in group were inoculated with pbs; piglets in group were inoculated with the sic of piglet in group with a dose of log pfu/ml targeting n gene of pedv. b fecal consistency (fc) was scored as follows: , solid, , pasty, , semiliquid; and , liquid. an fc score of and were considered diarrhea and severe diarrhea, respectively. c values in parentheses are the number of positive results/number of animals tested. d the detection limit of pedv rt-qpcr targeting the s gene of icpc a or icpc a-s Δ is log copies/ml. e the detection limit of pedv rt-qpcr targeting the n gene of both icpc a and icpc a-s Δ is . log copies/ml . f the pig number for diarrhea observation is or less than the total pig number because or pigs of each group were euthanized at hpi. g the experimental data were analyzed by student`s t-test. letters 'a, b and c' indicate a mean significant difference between groups (p < . ). '-', not detected. y. su et al. veterinary microbiology ( ) - at the tested concentrations had no effects on the infection of icpc a-s Δ (fig. b-e) . similarly, in ipec-dq cells, the infectivity of icpc a was increased significantly by . - . % bile but was decreased significantly by the concentration of . % (fig. f) . however, . - . % bile decreased the infectivity of icpc a-s Δ significantly. bile acids gcdca (fig. g-j) . no significant effects of bile acids on icpc a-s Δ were observed except for that the highest concentration ( μm) of gcdca and dca decreased its infectivity significantly (fig. g-j) . fig. . histopathological examination of the gn piglets infected with individual or both icpc a and icpc a-s Δ . (a) ihc staining of pedv n proteins in the jejunal and ileal sections of piglets that died or were euthanized at - dpi (magnification, ×) . the brown signals represented the pedv n antigens in enterocytes. (b) villous height to crypt depth (vh/cd) ratios of jejunum and ileum of the gn piglets euthanized at - dpi. for each intestinal section, villi and crypts were measured. data are shown as the m ± sd. number of asterisks indicate significant difference between groups (*, p < . ; **, p < . ; ***, p < . ). 'ns', not significant. (c) if staining of pedv s proteins in the jejunal sections of piglets that died or were euthanized at - dpi (magnification, x). tissue sections were stained for the detection of icpc a and icpc a-s Δ antigens targeting the s Δ protein (green), and for icpc a antigens targeting the s °domain (red), and counterstained for cell nuclei (blue). in the merged images, yellow dots represented icpc a-infected (or co-infected in coinfection group) cells (merged from red and green) and the green dots represented icpc a-s Δ infection alone. (magnification, x) . cells were stained for the detection of icpc a and icpc a-s Δ antigens targeting the s Δ protein (green), and for icpc a antigens targeting the s °domain (red), and counterstained for cell nuclei (blue). in the merged images, yellow dots represented icpc a-infected (or co-infected in coinfection condition) cells and the green dots represented icpc a-s Δ infection alone. the experimental data were analyzed by student`s t-test. letters 'a, b and c' indicate a mean significant difference between groups (p < . ). previously, our laboratory isolated the first s ntd-del pedv strain, pc , from vero cell culture (oka et al., ) . recent studies revealed that s ntd-del pedvs naturally evolved in the field and often coinfected pigs with the s-intact pedv (suzuki et al., ; diep et al., ; zhang et al., a; su et al., ) . the s ntd-del pedv had no tissue tropism change compared with the s-intact pedv (suzuki et al., ; hou et al., ) . this differs from porcine respiratory coronavirus (prcv) that is a s ntd-del version of tgev and changes the major tissue tropism from intestines to respiratory tract (zhang et al., ) . in this study, we investigated the replication of s ntd- fig. . infection of pedv icpc a and icpc a-s Δ in neuraminidase (na)-treated vero and ipec-dq cells. vero or ipec-dq cell monolayers in -well plates were treated with mu na or mock at ℃ for h. after three washings with dmem/rpmi- , cells were inoculated with equal amounts of virus at a moi of . diluted in the medium containing μg/ml trypsin. after incubation at ℃ for h, the inoculum was removed followed by three washings. dmem/rpmi- containing μg/ml soybean trypsin inhibitor (sbti) and swine pedv positive serum (virus neutralization titer : , : dilution) was added. at hpi, cells were fixed with acetone-methanol ( : ) and infectivity was tested by if assay. each experiment was performed three times. data are shown as the m ± sd, and number of asterisks indicate significant difference between groups (*, p < . ; **, p < . ; ***, p < . ). fig. . effect of bovine mucin (bm) and porcine gastric mucin (pgm) on the infection of pedv icpc a and icpc a-s Δ in vero (a and b) and ipec-dq cells (c and d). monolayers of vero or ipec-dq cells in -well plates were inoculated with a moi of . icpc a or icpc a-s Δ in medium containing μg/ml trypsin and different concentrations of bm or pgm. after incubation at ℃ for h, the inoculum was removed followed by three washings. dmem/rpmi- containing μg/ml soybean trypsin inhibitor (sbti) and swine pedv positive serum (virus neutralization titer : , : dilution) was added. at hpi, cells were fixed with acetone-methanol ( : ) and infectivity was tested by if assay. each experiment was performed three times. data are shown as m ± sd, and number of asterisks indicate significant difference between groups (*, p < . ; **, p < . ; ***, p < . ). del and s-intact pedvs during coinfection in pigs. we developed a duplex rt-qpcr targeting the s gene of pedv to differentiate the two viruses. we found that the duplex rt-qpcr assay had different sensitivities for the detection of infectious icpc a and icpc a-s Δ (table s ) although it had a similar detection limit ( log copies/ml) for both viruses. for example, log s gene copies/ ml corresponded to . and . log ffu/ml for icpc a and icpc a-s Δ , respectively. it may reflect the fact that icpc a-s Δ replicates to lower peak infectious titers (∼ . log ffu/ml) than that (∼ . log ffu/ml) of icpc a in vero cells although both viruses replicated to similar peak titers based on the s gene (∼ . log copies/ml) (fig. a) . in the mixture of two viruses, the detection fig. . effect of bile and bile acids on the infection of pedv icpc a and icpc a-s Δ in vero (a-e) and ipec-dq cells (f-j). monolayers of vero or ipec-dq cells in -well plates were inoculated with a moi of . icpc a or icpc a-s Δ in medium containing μg/ml trypsin and different concentrations of bile or bile acids. after incubation at ℃ for h, the inoculum was removed followed by three washings. dmem/rpmi- containing μg/ml sbti and swine pedv positive serum (virus neutralization titer : , : dilution) was added. at hpi, cells were fixed with acetone-methanol ( : ) and infectivity was tested by if assay. each experiment was performed three times. data are shown as m ± sd, and number of asterisks indicate significant difference between groups (*, p < . ; **, p < . ; ***, p < . ). sensitivity of each virus differed significantly (tables s ). for the detection of icpc a at high titers ( . log ffu/ml) and as the predominant virus in the mixture of two viruses (icpc a: icpc a-s Δ = - : ), the s gene titers (∼ . - . log s gene copies/ ml) (table s ) were similar to that (∼ . log s gene copies/ml) in the single virus alone (table s ). because icpc a replicated to high titers and was the predominant virus during the coinfection in pigs, we can predict that the peak infectious titer of icpc a in coinfection was ∼ . log -higher than that in its single infection based on the duplex rt-qpcr results (∼ . vs ∼ . log s gene copies/ml) (table ) . so, we conclude that the replication of icpc a was interfered at the beginning based on delayed timing ( . vs . dpi) to reach peak titers but enhanced eventually in coinfection compared with its single infection in pigs. on the other hand, the sensitivity for the detection of icpc a-s Δ decreased significantly and icpc a-s Δ became undetectable when icpc a was high ( . log ffu/ml) and icpc a-s Δ infectious titers were lower than . log ffu/ml in the mixture of two viruses (table s ). therefore, the decreased peak s gene titer (∼ . log copies/ml) of icpc a-s Δ in coinfection of pigs compared with that (∼ . log copies/ml) in its single infection may be due to inhibition of replication or the decreased detection sensitivity of the duplex rt-qpcr for the detection of icpc a-s Δ during coinfection (table ). in the first passage of coinfection, we observed much lower numbers of s ntd-del pedv-infected cells than the s-intact pedv-infected cells in the small intestines of pigs infected with the same dose of both viruses simultaneously. in addition, plaque assays were performed to isolate and purify individual viruses from the sic of one of the coinfection pigs. however, only s-intact pedv was isolated alone. in contrast, s ntd-del pedv was exclusively detected together with the sintact pedv from the plagues. in the second passage of the coinfection in pigs, s-intact pedv but not s ntd-del pedv rna was detected in the lic of the pig inoculated with the sic of the pig inoculated with both viruses. this finding suggests that s ntd-del pedv had no replication advantage and was either outcompeted or coexisted with sintact pedv in pigs. this conclusion is in agreement with what were observed in the field (diep et al., ; su et al., ) . s ntd-del pedv replicated to a lower peak titer in coinfection than that in single virus infection in both vero cells and ipec-dq cells. these in vitro results were similar to those in the pig studies. in the second passage of coinfection, s ntd-del pedv rna was detected in vero cells but not in ipec-dq cells, probably due to the lower replication efficiency of the s ntd-del pedv in ipec-dq cells than in vero cells ( fig. a and c) . similar to other sialic acid-binding coronaviruses, the s ntd of pedv has a sialic acid binding activity, and the sugar-binding activities of a field isolate pedv variant chgd- was stronger than that of the prototype strain pedv cv using bm (deng et al., ) . the sialic acid binding activity occurred within the n-terminal residues and the capacity of sialic acid binding differs among pedv strains using an hemagglutination assay . the recombinant virus used in this study, pedv icpc a-s Δ , is a s ntd-del version of icpc a. it lost most sialic acid binding activity tested in vero cells (hou et al., ) . in this study, we comparatively tested icpc a-s Δ and icpc a in vero and ipec-dq cells treated with na. similar to the previous reports, our results confirmed that the binding of s ntd of pedv to sialic acids on cell surface enhanced virus infectivity. bm is a mixture of highly glycosylated proteins containing sugar moieties, such as -n-acetyl- -o-acetylneuraminic acid (neu , ac ), -n-glycolylneuraminic acid (neu gc), and -n-acetylneuraminic acid (neu ac). among them, neu gc and neu ac can serve as receptors or co-receptors for some alphacoronaviruses (e.g., tgev) and gammacoronaviruses [e.g., infectious bronchitis virus (ibv)] (cavanagh and davis, ; krempl et al., ) . similar to tgev, which uses neu gc and neu ac as co-receptors (krempl et al., ; schultze et al., ; schwegmann-wessels and herrler, ) , the s ntd of pedv interacted with sugar (deng et al., ) . pgm contains . - . % n-acetylneuraminic acid (neuac). our data indicated that low-medium concentrations of bm or pgm enhanced the infectivity of s-intact pedv in both vero and ipec-dq cells. in contrast, the s ntd-del pedv was not affected or inhibited, probably due to the covered mucin that blocked virus binding to the receptors. the intestinal epithelial cells are covered by a layer of mucus that is rich in sialic acids. these results suggest that low-medium amount of mucin binding to s-intact pedv may help the virus particles approach the receptors via sticking to the sialic acids on the cell surface. however, the receptor binding domain (rbd) on the s protein of s ntd-del pedv, which lacks most sialic acid binding activity, is probably covered with mucin and blocked its binding to the receptors. when high concentration of mucin saturates the s ntd of s-intact pedv, it can also cover viral rbd and block its binding to the receptors, resulting in decreased infectivity as observed in . mg/ml bm effects on s-intact pedv in vero cells (fig. a) . the major components of bile include bile acids, cholesterol, lipids, bilirubin, proteins and carbohydrates. the bile acids are stored in the gall bladder (at ∼ mm) and released into the small intestine (mcleod and wiggins, ) . in the small intestine, the total bile acids have a concentration of - mm (dowling, ) . we selected two primary bile acids (gcdca and cdca) and two secondary bile acids (dca and udca) to test the effects of bile acids on pedv infection. the secondary bile acids are dehydroxylated ones from the primary bile acids by intestinal bacteria (björkhem, ) . our data indicated that a broad range of concentrations of bile and bile acids enhanced the infectivity of s-intact pedv in both vero and ipec-dq cells. however, bile and bile acids cannot increase the infectivity of s ntd-del pedv. these results suggest that the bile-and bile acid-mediated promotion of pedv infection is related to the s ntd. it was reported that gcdca may facilitate the adaptation of a s-intact pedv to trypsin-free growth in vero cells at the early passages (< p ) (kim et al., ) . however, the mechanisms of the bile acid function on the pedv infection is unknown and consequently need to be further investigated. the inability of the s ntd-del pedv to outcompete the s-intact pedv may account for the fact that s ntd-del pedvs were exclusively detected from coinfection with the s-intact pedv (diep et al., ; su et al., ) . consequently, the disease caused by the coinfection was as severe as that by the highly virulent s-intact pedv alone. this situation is quite different from what occurred for prcv and tgev: the wild spread of clinically mild prcv, which often infects pigs asymptomatically and repeatedly, inducing protective herd immunity against tgev outbreaks (vancott et al., ; brim et al., ) . prcv functions as a natural effective vaccine against tgev. however, for pedv, the clinically mild s ntd-del pedv variants do not infect pigs alone. therefore, safe and effective vaccines are still desired to control the deadly pedv infection in neonatal piglets. due to the % mortality rates of piglets in the icpc a-infected pigs (hou et al., ) and coinfection groups, we currently cannot determine whether the pathogenicity of pedv icpc a is enhanced during the coinfection. that should be investigated in older pigs (weaned pigs, sows and boars) which are more resistant to pedv infection and diseases (niederwerder and hesse, ) . in addition, the emergence of s ntd-del pedv variants may complicate pedv disease pattern on farms, which needs to be further investigated. in this study, we showed that the replication of the s-intact pedv was enhanced during coinfection with an s ntd-del pedv in pigs. we found that mucin, bile and bile acids can all increase the infection of sintact pedv but not the s ntd-del pedv. this feature may help explain why s-intact pedv outcompetes s ntd-del pedv in vivo. further studies are needed to understand the mechanisms of mucin-, bile-and bile acid-mediated enhancement or inhibition effects on the infection of different pedv variants. all animal experiments were performed according to the 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sequences, a key residue in the spike protein and deletions in nonstructural protein b of us strains of the virulent and attenuated coronaviruses, transmissible gastroenteritis virus and porcine respiratory coronavirus identification of porcine epidemic diarrhea virus variant with a large spike gene deletion from a clinical swine sample in the united states type iii interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp in irf signaling the authors declare no conflict of interest. none. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.vetmic. . . . key: cord- -h xn authors: araujo, danielle bastos; machado, rafael rahal guaragna; amgarten, deyvid emanuel; malta, fernanda de mello; de araujo, gabriel guarany; monteiro, cairo oliveira; candido, erika donizetti; soares, camila pereira; de menezes, fernando gatti; pires, ana carolina cornachioni; santana, rúbia anita ferraz; viana, amanda de oliveira; dorlass, erick; thomazelli, luciano; ferreira, luis carlos de sousa; botosso, viviane fongaro; carvalho, cristiane rodrigues guzzo; oliveira, danielle bruna leal; pinho, joão renato rebello; durigon, edison luiz title: sars-cov- isolation from the first reported patients in brazil and establishment of a coordinated task network date: - - journal: mem inst oswaldo cruz doi: . / - sha: doc_id: cord_uid: h xn background: severe acute respiratory syndrome coronavirus (sars-cov- ) was confirmed in brazil in february , the first cases were followed by an increase in the number of cases throughout the country, resulting in an important public health crisis that requires fast and coordinated responses. objectives: the objective of this work is to describe the isolation and propagation properties of sars-cov- isolates from the first confirmed cases of coronavirus disease (covid- ) in brazil. methods: after diagnosis in patients that returned from italy to the são paulo city in late february by rt-pcr, sars-cov- isolates were obtained in cell cultures and characterised by full genome sequencing, electron microscopy and in vitro replication properties. findings: the virus isolate was recovered from nasopharyngeal specimen, propagated in vero cells (e , ccl- and hslam), with clear cytopathic effects, and characterised by full genome sequencing, electron microscopy and in vitro replication properties. virus stocks - viable (titre . × ( ) tcid /ml, titre . × ( ) pfus/ml) and inactivated from isolate sars.cov /sp . .hiae.br were prepared and set available to the public health authorities and the scientific community in brazil and abroad. main conclusion: we believe that the protocols for virus growth and studies here described and the distribution initiative may constitute a viable model for other developing countries, not only to help a rapid effective pandemic response, but also to facilitate and support basic scientific research. mild upper respiratory tract disease with low mortality rates. ( ) however, in and , respectively, the emergence of highly pathogenic severe acute respiratory syndrome (sars-cov) ( ) and middle east respiratory syndrome (mers-cov) ( ) revealed that this virus group may also cause severe respiratory illness in humans. in december , in wuhan, china, a novel coronavirus, member of the β coronavirus family, has been identified as the source of a pneumonia outbreak ( ) and this novel virus was named as severe acute respiratory syndrome coronavirus (sars-cov- ), by the international committee on taxonomy of viruses (ictv). ( ) in brazil, the four endemic covs circulate annually ( ) ( ) ( ) ( ) ( ) ( ) and the first case of coronavirus disease (covid- ) was reported on february , (https://covid.saude. gov.br) when sars-cov- was detected in a -year-old male traveller from lombardia region, italy, that returned to the são paulo city, brazil. until the first reported case in brazil, also the first in south american region, ( ) there were , confirmed covid- cases in countries. after these first reported patient, cases in brazil started to rise reaching , , infected persons and , deaths on august (https://covid.saude.gov.br). isolation and propagation of new viruses in vitro represents an essential step and may generate important primary tools in early outbreak characterisation. in that way, isolation of the virus presently disseminating in brazil will provide important information regarding diversity and molecular evolution of the pathogen, but also supply reference material in the struggle to control the rampant spreading of the pandemic sars-cov- in the country. indeed, the availability of infective particles as well as inactivated genetic material as reference reagents are extremely necessary for the preparation of positive controls in molecular diagnosis, development of vaccine formulations, detection of neutralising antibodies, screening of antiviral compounds and for different basic research projects both for public health reference laboratories and the research community. in this study, we describe the isolation of sars-cov- from the first two patients diagnosed with the novel coronavirus disease in brazil. we describe its genomic sequence (sars-cov- /sp /human/ /bra) and in vitro replication characteristics. virus stocks (infectious particles and lysates) were set available and distributed to the research community. ethics declarations -all methods were performed in accordance with relevant guidelines and regulations. this work was approved by the ethics committee on research with humans from the institute of biomedical sciences, university of são paulo, brazil (permission number . . . ). all specimens were handled under the laboratory biosafety guidance required for the novel coronavirus ( -ncov) by the world health organization (who) ( ) at bls facilities at the institute of biomedical sciences, university of são paulo. clinical specimen collection -nasopharyngeal (np) swab samples were collected from symptomatic patients who had acquired covid- during travels to northwest of italy (lombardia region) and returned to the são paulo city in late february. these patients were treated in the same hospital and were the two first confirmed cases of covid- in the são paulo city. the specimens were collected on day - post-symptom onset, placed in - ml of saline medium and used for molecular diagnosis and virus isolation. nucleic acid extraction and real-time rt-qpcr for virus detection -in order to perform the identification of sars-cov- , the extraction of total nucleic acid (rna and dna) from the collected samples ( µl of initial material) were carried out using the semi-automated nuclisens ® easymag ® platform (biomérieux, lyon, france), following the manufacturer's' instructions. all specimens were handled under the laboratory biosafety guidance required for the novel coronavirus ( -ncov) by who ( ) at bls facilities at the institute of biomedical sciences, university of são paulo. the de-tection of viral rna was carried out using the agpath-id one-step rt-pcr kit (applied biosystems inc., waltham, usa) on an abi sds real-time pcr machine (applied biosystems, weiterstadt, germany), using a published protocol and sequence of primers and probe for e gene. ( ) rna copies/ml was quantified by real-time rt-qpcr using a specific in vitro-transcribed rna quantification standard, kindly granted by christian drosten, charité -universitätsmedizin berlin, germany, as described previously. ( ) virus isolation -we used vero e cells for isolation and initial passages. we cultured vero e in dulbecco minimal essential medium (dmem) supplemented with % of heat-inactivated foetal bovine serum (fbs) (vitrocell embriolife, campinas, brazil). we used np swab specimen for virus isolation. for isolation and first passage, we sow cells in a cm cell culture flask in a concentration of × cells/ml. after h, we removed the culture medium, washed three times with fbs free-dmem and inoculated aliquots ( μl) of the clinical specimens into the flask. after h of incubation (adsorption), we completed the volume for ml with dmem supplemented with . % fbs and % of penicillin-streptomycin. we grew the inoculated cultures in a humidified °c incubator in an atmosphere of % co and observed for cytopathic effects (cpe) daily up to h. supernatant was collected daily, and virus replication was confirmed through cpe, gene detection and electron microscopy. virus titration -median tissue culture infectious dose (tcid /ml) -vero e and ccl- cells were seeded into -well plate ( × cells/ml), h before the experiment. virus was -fold serially diluted in medium ( - to - ). medium was removed from plates, virus dilutions applied in sextuplicate and incubated at °c. visualisations were performed daily in an inverted light microscope (axiovert , carl zeiss oberkochen) to observe the cpe. after h, the last reading was performed, and the monolayers were fixed and stained with naphthol blue black (sigma-aldrich co., deisenhofen, germany) dissolved in sodium acetate-acid acetic. the viral titre was expressed in tcid /ml and calculated using the spearman & kärber algorithm, as described by hierholzer & killington. ( ) plaque forming units (pfu/ml) -virus titration was carried out in wells plates seeded with vero e and ccl- cells at a concentration of × cells/well. after h and a cell confluence of - %, dilutions - to - in dmem . % fbs of the virus was transferred in duplicate ( µl/well) to the seeded plates. after h adsorption at o c % co , the wells were completed with an overlay of carboxymethyl cellulose (cmc) with dmem, % fbs and % of penicillin-streptomycin, and plates incubated at o c in % co and stained with naphtol blue black dissolved in sodium acetate-acid acetic. plates were observed and stained from to h post-inoculation (h.p.i.). both virus titration (tcid / ml and pfu/ml) were made after the third passage of the isolated virus (t ). samples were adsorbed to glow-discharged carbon filmcoated copper grids ( mesh, cf -cu, electron microscopy sciences). the grids were washed with ultrapure water treated with depc and negatively stained with uranyl acetate % (w/v) with blotting on filter paper after each step. a fei tecnai g kv transmission electron microscope (department of cell and developmental biology, institute of biomedical sciences, university of são paulo) was used for image acquisition. virus growth kinetics in different cell lines -three vero cell lines (e , ccl- and hslam) plus a human epithelial type (hep- ) cells, at concentration of × cells/ml, were tested for the propagation of the sars-cov- by inoculation at a multiplicity of infection (moi) of . . the culture medium consisted of dmem supplemented with . % of fbs. aliquots of cell-associated and supernatants compartments were collected every h up to h.p.i. for virus quantification via tcid / ml and rna copy number quantification by reverse transcription-quantitative polymerase chain reaction (rt-qpcr). the assay was conducted in triplicate, reproduced in two independent experiments and expressed by standard error of the mean (sem). graphics and sem were done using graphpad prism software version . (graphpad software, san diego, usa). next generation sequencing of viral full-length genome -we extracted total nucleic acid from the np and oropharyngeal (op) swab samples and cell supernatants isolates with the qiaamp viral rna mini kit (qiagen, hilden, germany). the purification and concentration steps were carried out with rna clean & concentrator kit (zymo research, irvine, usa) with dnase i treatment during the concentration process. depletion of human ribosomal rna was performed with the concentrated rna product using the qiaseq fast select rna removal kit (qiagen). finally, the rna samples were submitted to random amplification following the methodology described in greninger et al. ( ) with few modifications. the preparation of sequencing libraries for the illumina platform was carried out with the nextera xt kit (illumina, san diego, usa) and multiplex testing, using the random two-step pcr amplification product as input, followed the kit's standard instructions. the libraries were quantified after fluorescence measuring with the qubit instrument (thermo fisher scientific, waltham, usa) and loaded on the nextseq equipment (illumina) for sequencing with mid pairedend reads (illumina). sequencing analysis -the sequencing data was analysed by a flow of bioinformatics analysis (pipeline) developed at albert einstein hospital. in summary, raw sequencing data was subjected to sequence quality controls, removal of human contaminants by aligning against the hg reference genome, taxonomic identification of other pathogens and genome recovery through manual curing. quality control was performed using cutadapt ( ) to filter sequences by length (< bp), average quality (q p < ) and trim options to remove low quality ends ( bp to ' end and bp to ' end). passed qc reads were mapped to hg human reference genome using bwa ( ) mem with default parameters. not mapped reads were submitted to assembly using spades . . ( ) contigs were inspected and manually curated using geneious . to generate a final assembly. complete genome was compared to sars-cov- reference and close isolates by multiple sequence alignment. final genome was deposited in genbank (https:// www.ncbi.nlm.nih.gov/genbank/). the preparation of vis and vls stocks was performed as described above for virus isolation. clinical specimen collection -patient (hiae ), a -years-old male patient, and patient (hiae ), a -years-old male patient, had returned from northwest of italy (lombardy region) and presented respiratory symptoms including cough, sore throat, runny nose, fever, myalgia and headache. patient was initially diagnosed with a community-acquired pneumonia and received antimicrobial therapy. both were confirmed for covid- by hospital israelita albert einstein (hiae), in the são paulo city on february (hiae ) and (hiae ), . a summary of clinical characteristics of the patients is described in table. lombardy is considered the centre of the covid- outbreak in italy ( ) and has a high influence of the first wave of sars-cov- introduced in brazil. ( ) virus isolation -before isolation in cell cultures, we tested the samples using a one-step multiplex rt-qpcr for the detection of additional different respiratory viruses ( ) and tested for bacterial contamination using fluid thioglycolate medium (becton dickinson, franklin lakes, usa). no other pathogens were detected. the positive np were inoculated on vero e cells. the initial sample collected from hiae was freezed and thawed before inoculation and no virus propagation was obtained. a second sample, from the same patient, was obtained "fresh" (conserved at ºc for no longer than h) and we could successfully isolate sars-cov- . sample from hiae was inoculated "fresh" from the first moment. the failure to isolate the virus from the first sample collected from hiae may be attributed to a lower virus load and to the freeze-thaw cycle before cell culture inoculation. the schematic timeline of procedures is presented at fig. . three days post infection, the isolation of sars-cov- from hiae was confirmed by rt-qpcr, electron microscopy and whole genome sequencing (fig. ) . the cycle threshold (ct) value and genome copy numbers (rna copies/ml) of the pre-inoculated sample was ct . and . × (fig. a) . rna quantification of passages and after h.p.i. gave values of . - . × copies/ml and . - . × copies/ml, respectively. since virus isolation from hiae (sars.cov /sp . .hiae.br) was faster, all the subsequent studies were carried out with this isolate after passage . virus isolation (passage ) from hiae (sars.cov /sp . .hiae.br) was confirmed by rt-qpcr (ct . - . × copies/ml) and whole genome sequencing, being stored at - ºc. negative staining transmission electron microscopy of the sars.cov /sp . .hiae.br, here after referred as sp /bra, permitted the observation of coronavirus-specific morphological structure, being possible to visualise the protein components of the viral envelope. the virus particle size ranged from to nm (fig. b ). the cpes were characteristic of sars-cov- : cell rounding, detachment of the cell monolayer and formation of loose cells on the surface, for both vero e and ccl- cells and similar to previously observed effects. ( ) ( ) ( ) ( ) nonetheless, the cpes were more evident in ccl- cells. the virus isolate was titrated after two blind passages following isolation (t ) and the cpes were more clearly observed in ccl- cells for tcid /ml ( . × ) and pfus ( . × ). for pfus titration, effects were not clear for vero e cells, and for vero ccl , only a few small plaques were visible at h.p.i., being much larger and more visible at h.p.i. (supplementary data, fig. ). hartcourt et al. ( ) described effects more visible for vero e cells when compared to ccl- . other successful sars-cov- isolation reports were based on vero e , vero ccl- and vero hslam ( ) ( ) ( ) ( ) ( ) showing that the three cell lines are permissive for sars-cov- . virus growth kinetics in different cell lines -we examined sars-cov- growth kinetics in three vero cell lines (e , ccl- and hslam) and compared with hep- cells. all cells were inoculated with the sars-cov- isolate (sp /bra) and cultured under similar conditions. virus titre quantification analysis of cell-associated and supernatants compartments indicated that similar levels of infectious sars-cov- were produced in all three vero cell lines of vero, but not in hep- cells, that proved to be not permissive to virus replication. the peak of viral titre was detected h.p.i. ( tcid /ml) after an initial eclipse phase. cpes was not observed until h.p.i. and reached a peak at h.p.i. (supplementary data, fig. ). quantification of ribonucleic acid copy number (rna cn ) indicated that virus was released into the supernatant with similar kinetics for all three tested vero cell lines, with virus yielding slightly higher values on h.p.i. (fig. b) . rnacp:tcid ratios did not differ significantly (p > . ) among the tested vero cell lines (fig. c ). in addition, these cell lines appeared to release few noninfectious particles at early time points of the infection. the rnacp:tcid ratios appeared to increase discreetly over time, suggesting an increase in the release of noninfectious virus at later time points or an increase in virus particle degradation over time (perhaps as a consequence of cell culture proteases). virus rna was not detected in the cell-associated fraction and cell cultures supernatants of hep- cells (fig. a , b). any clear-characteristic cpe of sars-cov- was observed in hep- cells (supplementary data, fig. ) . similarly to other studies with sars-cov ( , ) and sars-cov- , ( , ) our findings demonstrated that the three tested vero cell lines release infectious virus particles and viral rna copies at the same kinetics and efficient egress. whole genome sequencing -whole genome sequence of the sars-cov- wuhan-hu- (nc_ ) and inmi /ita (mt ) were compared with sp / bra directly isolated from patient's sample (mt ) and after cultivation in vero cells (mt ) using mafft multiple aligner tool (algorithm = auto and pam = ). alignment shows only two mutations at the cultivated strain (> . % similarity). mutations occurred at the nsp and spike proteins (fig. ) . distribution network -until march , , the sending of the material comprised different research groups, in public and particular university/hospitals, at different states in brazil (fig. ) . the inactivated virus (vls) was distributed according of request from the laboratories to testing clinical samples by rt-qpcr, using the vls as positive controls, which was important, considering the difficulties -availability and price -to import synthetic rna in brazil. the criteria for distributing live virus (vis) was based in the capacity of bsl facilities from the host institutions, experience of the principal investigator and the analysis of priority for development of vaccine, drug discovery and virus neutralisation diagnosis. this initiative is crucial to improve the study of sars-cov- and the development of methods and strategies for virus treatment and prevention. sars- cov- isolates were set available to the scientific community by different groups in other countries. ( , ) the first delivery to the research community at the são paulo state was set on march . the virus distribution by the brazilian mail company was first set on march , and, in less than h, the biological material was delivered to rio de janeiro ( . miles away from the são paulo city), the state of minas gerais ( . miles) and the state of rio grande do sul ( . km). virus samples were sent following technical and biosafety requirements, in accordance with anvisa recommendations. in conclusion, in this work, we describe the successful isolation of sars-cov- from the first diagnosed patients in brazil. the virus was propagated in vero cell lines and replication features, cpe and growth kinetics were described. the experimental protocols described herein can be used for future attempts to isolate sars-cov- in different places in the world. the produced virus stocks were distributed to different research groups and hospitals in the country and are being used as a reference in diagnostic tests and for research, aiming the screening of antivirus drugs, testing the efficacy of vaccine formulations under experimental conditions. the vls are been used as controls for molecular diagnosis and studies, eliminating the need of imported material in the first weeks of the pandemic in brazil. the covid- pandemic is unprecedent and the collaborative work is crucial to the efforts to understand and control the virus spread in the country. data availability -the complete genome sequences of sars-cov- /sp /human/ /bra from de clinical sample and sars-cov- /human/bra/ sp cc/ from cell culture isolation have been deposited in the genbank (accession mt and mt , respectively). the version described in this paper is the first version. epidemiology, genetic recombination, and pathogenesis of coronaviruses identification of a novel coronavirus in patients with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia a novel coronavirus 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real-time nanopore sequencing analysis cutadapt removes adapter sequences from highthroughput sequencing reads fast and accurate short read alignment with burrows-wheeler transform spades: a new genome assembly algorithm and its applications to single-cell sequencing spread and dynamics of the covid- epidemic in italy: effects of emergency containment measures routes for covid- importation in brazil comparison of fast-track diagnostics respiratory pathogens multiplex real-time rt-pcr assay with in-house singleplex assays for comprehensive detection of human respiratory viruses severe acute respiratory syndrome coronavirus from patient with novel coronavirus disease, united states virus isolation from the first patient with sars-cov- in korea serological and molecular findings during sars-cov- infection: the first case study in finland comparative tropism, replication kinetics, and cell damage profiling of sars-cov- and sars-cov with implications for clinical manifestations, transmissibility, and laboratory studies of covid- : an observational study isolation and rapid sharing of novel coronavirus (sars-cov- ) from the first patient diagnosed with covid- in australia sars-associated coronavirus replication in cell lines discovery of novel human and animal cells infected by the severe acute respiratory syndrome coronavirus by replicationspecific multiplex reverse transcription-pcr to roberto cabado, for technical assistance at the electron microscopy facilities at department of cell and developmental biology, institute of biomedical sciences, university of são paulo, to priscila perine, for viral purification and concentration, to the lvcm covid- working group members (bruna larotonda telezynski, camila araujo valério, fabyano bruno leal, ralyria mello and vanessa nascimento chalup), and to the brazilian mail company and the mctic of brazil, for the logistic support in virus distribution. key: cord- -nbgdmavr authors: kim, youngnam; lee, changhee title: ribavirin efficiently suppresses porcine nidovirus replication date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: nbgdmavr porcine reproductive and respiratory syndrome virus (prrsv) and porcine epidemic diarrhea virus (pedv) are porcine nidoviruses that represent emerging viral pathogens causing heavy economic impacts on the swine industry. although ribavirin is a well-known antiviral drug against a broad range of both dna and rna viruses in vitro, its inhibitory effect and mechanism of action on porcine nidovirus replication remains to be elucidated. therefore, the present study was conducted to determine whether ribavirin suppresses porcine nidovirus infection. our results demonstrated that ribavirin treatment dose-dependently inhibited the replication of both nidoviruses. the antiviral activity of ribavirin on porcine nidovirus replication was found to be primarily exerted at early times post-infection. treatment with ribavirin resulted in marked reduction of viral genomic and subgenomic rna synthesis, viral protein expression, and progeny virus production in a dose-dependent manner. investigations into the mechanism of action of ribavirin against prrsv and pedv revealed that the addition of guanosine to the ribavirin treatment significantly reversed the antiviral effects, suggesting that depletion of the intracellular gtp pool by inhibiting imp dehydrogenase may be essential for ribavirin activity. further sequencing analysis showed that the mutation frequency in ribavirin-treated cells was similar to that in untreated cells, indicating that ribavirin did not induce error-prone replication. taken together, our data indicate that ribavirin might not only be a good therapeutic agent against porcine nidovirus, but also a potential candidate to be evaluated against other human and animal coronaviruses. the nidovirales are an order of enveloped single-stranded positive-sense rna viruses with animal hosts that include the families arteriviridae, coronaviridae, and roniviridae (cavanagh, ; mayo, ; spaan et al., ) . despite striking differences in genome size and virion morphology, the genome organization and expression strategy of the two groups belonging to the nidovirales order were found to be comparable. the nidovirus genome contains two large orfs, a and b, comprising the two-thirds of the viral genome encoding non-structural proteins (nsps) and the remaining orfs located in the terminal region coding for structural proteins (lai et al., ; snijder and spaan, ) . the initial translation from orf a and orf b yields the a and lab replicase polyproteins, respectively, which are then proteolytically processed into functional nsps including the viral rna-dependent rna polymerase (rdrp) (bautista et al., ; van aken et al., ; ziebuhr et al., ) . the rdrp-containing replication complex mediates genomic rna replication and subgenomic (sg) mrna transcription, eventually generating a nested set of -coterminal sg mrnas that are individually translated to structural proteins (lai et al., ; snijder and spaan, ) . porcine reproductive and respiratory syndrome virus (prrsv), a pathogenic macrophage-tropic arterivirus of pigs, is the etiological agent of acute respiratory illness in young piglets and reproductive failure in pregnant sows (albina, ) . prrsv primarily replicates in porcine alveolar macrophages (pams) and can establish persistent infection in lymphoid tissues of infected pigs that lasts for several months (albina et al., ; christopher-hennings et al., ; duan et al., ; wills et al., ) . as a result, prrsv infection results in suppression of normal macrophage functions and immune responses, which may render pigs susceptible to secondary bacterial or viral infections, leading to more severe disease than either agent alone (allan et al., ; feng et al., ; harms et al., ; wills et al., ) . porcine epidemic diarrhea virus (pedv), a pathogenic enterocyte-tropic coronavirus of swine, is the etiological agent of acute enteritis, which is characterized by lethal watery diarrhea followed by dehydration leading to death with a high mortality rate in suckling piglets (debouck and pensaert, ) . these two viruses, prrsv and pedv, are devastating porcine nidoviral pathogens that have still continued to plague swineproducing nations, causing tremendous economic losses to the global and asian pork industries (neumann et al., ; pensaert and yeo, ) . ribavirin ( -␤-d-ribofuranosyl- , , -triazole- -carboxamide, also known as virazole) is a synthetic guanosine analog that exhibits broad-spectrum antiviral activity in vitro (sidwell et al., ) . it has been used experimentally against a wide range of both dna and rna viruses, including gb virus b, hantaan virus, hendra virus, respiratory syncytial virus, lassa fever virus, norwalk virus, and west nile virus (chang and george, ; cooper et al., ; day et al., ; mccormick et al., ; lanford et al., ; rockx et al., ; severson et al., ) . most notably, ribavirin is used in combination with interferon-␣ for treatment of chronic hepatitis c virus (hcv) infections (cummings et al., ; davis et al., ) . however, there is still no report regarding an antiviral effect of ribavirin during the replication cycle of porcine nidoviruses. in the present study, therefore, we tried to investigate the antiviral activity of ribavirin and its mechanism of action in target cells upon porcine nidovirus infection. independent treatment of target cells with ribavirin significantly impaired prrsv and pedv infection. further experiments revealed that suppression of ribavirin affects post-entry steps of the replication cycle of prrsv and pedv, including viral genomic and sg rna synthesis, viral protein expression, and virus production. the addition of guanosine to the ribavirin treatment resulted in moderate reversal of the antiviral effects, suggesting that ribavirin activity is involved in the depression of cellular guanosine triphosphate (gtp) levels. sequencing analysis of the prrsv and pedv genomes in the ribavirin-treated and non-treated groups revealed that the mutation rates were similar and indicated that ribavirin did not induce catastrophic mutations during the replication of porcine nidoviruses. altogether, our results suggest that ribavirin may be an excellent therapeutic option for nidovirus infection in a human or veterinary subject. pam-pcd cells were cultured in rpmi medium (invitrogen, carlsbad, ca) supplemented with % fetal bovine serum (fbs, invitrogen), antibiotic-antimycotic solutions ( ×; invitrogen), mm hepes (invitrogen), mm sodium pyruvate (invitrogen), and nonessential amino acids ( ×; invitrogen) in the presence of g/ml zeocin (invitrogen). vero cells were cultured in alpha minimum essential medium (␣-mem, invitrogen) with % fbs and antibiotic-antimycotic solutions. the cells were maintained at • c in a humidified % co incubator. prrsv strain vr- was propagated in pam-pcd cells as described previously . pedv strain sm - was kindly provided by the korean animal, plant and fisheries quarantine and inspection agency and propagated in vero cells as described previously (hofmann and wyler, ) . ribavirin and mycophenolic acid (mpa) were purchased from sigma (st. louis, mo) and dissolved in distilled water (dw) or dimethyl sulfoxide (dmso), respectively. a monoclonal antibody (mab; sdow ) against the prrsv n protein was purchased from rural technologies (brookings, sd). the pedv spike (s) glycoprotein-specific and n protein-specific monoclonal antibodies (mabs) were kind gifts from sang-geon yeo (kyungpook national university, daegu, south korea). the anti-␤-actin antibody and horseradish peroxidase (hrp)-conjugated secondary antibody were purchased from santa cruz biotechnology (santa cruz, ca). the cytotoxic effects of reagents on pam-pcd and vero cells were analyzed by a -( , -dimethylthiazol- -yl)- , diphenyltetrazolium bromide (mtt) assay (sigma) detecting cell viability. briefly, pam-pcd and vero cells were grown at × cells/well in a -well tissue culture plate with ribavirin or mpa treatment for h. after one day of incubation, l of mtt solution ( . mg/ml) was added to each well and the samples were incubated for an additional h. the supernatant was then removed from each well, after which l of dmso was added to dissolve the color formazan crystal produced from the mtt. the absorbance of the solution was measured at nm using an enzyme-linked immunosorbent assay plate reader. all mtt assays were performed in triplicate. pam-pcd and vero cells grown on microscope coverslips placed in -well tissue culture plates were pretreated with ribavirin or mpa for h and mock infected or infected with prrsv and pedv at a multiplicity of infection (moi) of , respectively. the virusinfected cells were further grown in the presence of ribavirin until hpi, fixed with % paraformaldehyde for min at room temperature (rt) and permeabilized with . % triton x- in pbs at rt for min. the cells were blocked with % bovine serum albumin (bsa) in pbs for min at rt and then incubated with n-specific mab for h. after being washed five times in pbs, the cells were incubated for h at rt with a goat anti-mouse secondary antibody conjugated with alexa fluor (molecular probes, carlsbad, ca), followed by counterstaining with , -diamidino- -phenylindole (dapi; sigma). the coverslips were mounted on microscope glass slides in mounting buffer ( % glycerol and . % sodium azide in pbs) and cell staining was visualized using a fluorescent leica dm il led microscope (leica, wetzlar, germany). pam-pcd and vero cells were grown in -well tissue culture plates for day and were mock infected or infected with prrsv or pedv at an moi of . . at the indicated times, cells were harvested in l of lysis buffer ( . % tritonx- , mm ␤glycerophosphate, mm -nitro phenyl phosphate, mm mops, mm, mgcl , mm nacl, mm egta [ph . ], mm sodium orthovanadate, g/ml e , g/ml aprotinin, g/ml leupeptin, and mm pmsf) and sonicated on ice five times for s each. homogenates were lysed for min on ice, and clarified by centrifugation at , × g (eppendorf centrifuge r, hamburg, germany) for min at • c. the protein concentrations of the cell lysates were determined by a bca protein assay (pierce, rockford, il). the cell lysates were mixed with × nupage sample buffer (invitrogen) and boiled at • c for min. the proteins were separated on nupage - % gradient bis-tris gel (invitrogen) under reducing conditions, and electrotransferred onto immunobilon-p (millipore, billerica, ma). the membranes were subsequently blocked with % powdered skim milk (bd biosciences, belford, ma) in tbs ( mm tris-hcl [ph . ], mm nacl) with . % tween- (tbst) at • c for h, and reacted at • c overnight with the primary antibody against prrsv n, pedv s or ␤-actin. the blots were then incubated with the secondary horseradish peroxidase (hrp)-labeled antibody (santa cruz biotechnology) at a dilution of : for h at • c. proteins were visualized by enhanced chemiluminescence (ecl) reagents (amersham biosciences, piscataway, nj) according to the manufacturer's instructions. to quantify viral protein production, band densities of prrsv n and pedv s proteins were quantitatively analyzed using a computer densitometer with the wright cell imaging facility (wcif) version of the imagej software package (http://www.uhnresearch.ca/facilities/wcif/imagej/) based on the density value relative to ␤-actin gene. pam-pcd and vero cells were infected with prrsv or pedv, respectively, at an moi of . as described above. at − , , , , , , , , , or hpi, ribavirin was added to give the indicated final concentration for the remainder of the time course experiment. the virus-infected and ribavirin-treated cells were further maintained and the infection was terminated at hpi by fixing the monolayers with % paraformaldehyde for min at rt. the fixed cells were subjected to ifa to assess the presence of prrsv or pedv infection as described above. an internalization assay was performed as described previously (cai et al., ) . briefly, pam-pcd and vero cells grown in -well culture plates were infected with prrsv and pedv, respectively, at an moi of . at • c for h, respectively. unbound viruses were then washed with pbs and the cells were either incubated at • c or • c in the presence ribavirin for h, followed by treatment with protease k ( . mg/ml) at • c for min to remove the bound but uninternalized virus particles. the prrsv-infected cells were then serially diluted in rpmi and added onto fresh pam-pcd cell monolayers in -well tissue culture plates. at h post-incubation, internalized viruses were titrated through ifa as described above and the % tissue culture infectious dose (tcid ) was calculated. for pedv, the serially diluted infected cells were added onto uninfected vero cells and after h, internalized viruses were titrated using a plaque assay as described previously , and the plaques were counted after % crystal violet staining. pam-pcd and vero cells were incubated with ribavirin for h prior to infection and then inoculated with prrsv or pedv at an moi of for h at • c. the virus inoculum was subsequently removed and the infected cells were maintained in fresh medium containing ribavirin for h. total rna was extracted from lysates of the infected cells at hpi using trizol reagent (invitrogen) and treated with dnase i (takara) according to the manufacturer's protocols. the concentrations of the extracted rna were measured using a nanovue spectrophotometer (ge healthcare, piscataway, nj). quantitative real-time rt-pcr was conducted using a thermal cycler dice real time system (takara) with gene-specific primer sets (table ) as described previously (sagong and lee, ) . the rna levels of viral genes were normalized to that of mrna for the ␤-actin or glyceraldehyde- -phosphate dehydrogenase (gapdh) gene and relative quantities (rq) of mrna accumulation were evaluated using the − ct method. to detect alteration of genomic rna and sg mrna levels in the presence of ribavirin during porcine nidovirus infection, the results obtained using ribavirin-treated samples were compared with vehicle-treated results. pam-pcd and vero cells were prrsv or pedv infected with treatment of ribavirin as described above. the culture supernatant was collected at different time points ( , , , , and hpi) and stored at − • c. the titer of prrsv was measured by limiting dilution on pam-pcd cells through ifa as described above and then quantified as the tcid per ml. the pedv titer was determined by a plaque assay using vero cells and quantified as plaque-forming units (pfu) per ml. pam-pcd and vero cells were preincubated with m guanosine for h before ribavirin was added to the medium at various final concentrations and then inoculated with prrsv or pedv as described above. the virus inoculum was removed and the infected cells were maintained in fresh medium containing ribavirin and guanosine. at h dpi, the virus-infected cells were fixed and subjected to ifa to evaluate the presence of prrsv or pedv infection as described above. the n gene coding regions for prrsv and pedv were sequenced to monitor mutation frequency. cells were prrsv or pedv infected and treated with ribavirin as described above. at hpi, total rna was extracted by trizol, and rt-pcr was performed to amplify the region encoding the orf to orf genes of prrsv or the n gene of pedv using gene-specific primer sets (table ) . the corresponding pcr product was then cloned into the pgem-t vector (promega, madison, wi). for sequencing of the gene in the recombinant vector, we selected independent bacterial colonies per group and analyzed mutations in the region. a student's t test was used for all statistical analyses and pvalues of less than . were considered statistically significant. virus-specific cpes were observed daily and were photographed at hpi using an inverted microscope at a magnification of × (first panels). for immunostaining, infected cells were fixed at hpi and incubated with mab against the n protein of prrsv or pedv followed by alexa green-conjugated goat anti-mouse secondary antibody (second panels). the cells were then counterstained with dapi (third panels) and examined using a fluorescent microscope at × magnification. viral productions in the presence of ribavirin were measured by quantifying the number of cells expressing n proteins through ifa. five fields at × magnification were counted per each condition and the total number of cells per field as determined by dapi staining was similar in all fields. values are representative of the mean of three independent experiments and error bars represent standard deviations. *p = . - . ; † p < . . to examine the effect of ribavirin on porcine nidovirus replication, prrsv and pedv were selected because they are important viral pathogens that economically affect the swine industry. based on mtt assay, none of the doses of ribavirin tested in the present study caused detectable cell death of pam-pcd and vero cells (data not shown). pam-pcd and vero cells were pretreated with ribavirin at concentrations of - m or - m, respectively, or with dw as a vehicle control for h prior to infection. ribavirin was present during the entire period of infection. viral production was initially measured by monitoring the cytopathic effect (cpe) after infection and confirmed by immunofluorescence using n protein-specific mab at hpi (fig. ) . in vehicle-treated control cells, visible cpe appeared at hpi and became predominant by hpi, and n-specific staining was clearly evident in many cell clusters, indicating infection and the spread of the virus to neighboring cells. in contrast, ribavirin had an obvious inhibitory effect on virus propagation. as shown in fig. a and b, ribavirin significantly decreased virus-induced cpe and viral gene expression of prrsv and pedv at concentrations of ∼ m and ∼ m, respectively. n protein staining revealed that the number of cells expressing viral antigen, as quantified by n protein staining results, was also reduced during ribavirin treatment, resulting in a maximum of ∼ % inhibition in response to m and m for prrsv and pedv, respectively ( fig. a and b) . moreover, the effective doses for inhibiting % (ed ) of the replication of prrsv and pedv were determined to be about m and m, respectively. taken together, these data demonstrate that ribavirin efficiently suppresses the replication of porcine nidoviruses. to further determine at which point ribavirin acted during the porcine nidovirus infection period, pam-pcd and vero cells were treated independently with ribavirin at various time points post-infection. at hpi, the levels of prrsv or pedv replication were measured indirectly as viral antigen production by quantifying cells expressing the n protein through ifa (fig. ) . treatment of cells with m ribavirin for up to hpi resulted in more than - % decrease in prrsv production when compared to the untreated control. addition of ribavirin between and hpi led to - % inhibition of prrsv infectivity. similarly, treatment with m ribavirin at − , , and hpi was found to lead to - % suppression of pedv production, while its treatment at - hpi resulted in - % reduction in pedv infectivity. in contrast, no significant impairment of porcine nidovirus propagation was observed when ribavirin was added at hpi. these results demonstrate that the inhibitory effect of ribavirin against the replication of prrsv and pedv was exerted primarily during the initial period of infection, suggesting that its action occurs mainly at early time points after porcine nidovirus infection. to further assess the antiviral activity of ribavirin against porcine nidovirus replication, virus yield was determined during treatment of ribavirin. upon infection, viral supernatants were collected at hpi and viral titers were measured. as fig. a shows, the presence of ribavirin reduced the release of viral progeny in a dose-dependent manner. the peak viral titer was determined to be tcid /ml and . pfu/ml in the vehicle-treated control for prrsv and pedv, respectively. however, the addition of m or m ribavirin decreased titers of prrsv and pedv to . tcid /ml and . pfu/ml, respectively (almost log reduction compared with the control). the growth kinetics study further demonstrated that the overall process of porcine nidovirus replication was significantly delayed when cells were treated with ribavirin (fig. b) . consequently, these findings revealed that ribavirin inhibits optimal progeny virus release within the natural host cells. to evaluate which steps of the replication cycle of porcine nidoviruses were targeted by ribavirin, we started focusing on the earliest step, virus entry upon the ribavirin treatment. to address this issue, an internalization assay was performed as described previously (cai et al., ) . pam-pcd and vero cells were inoculated with prrsv and pedv, respectively, at • c for h to allow virus attachment and further maintained either at • c or • c to permit virus internalization in the presence of ribavirin. the samples were then treated with protease k to remove the remaining viruses from the cell surface. the serially diluted infected cells were subsequently subjected to an infectious center assay on uninfected pam-pcd and vero cell monolayers and virus titers were measured days later through ifa or by plaque assay. as shown in fig. c , the titers of prrsv and pedv were virtually the same in cells treated with ribavirin or without ribavirin at • c and determined to be . - . tcid /ml and . × - . × pfu/ml, respectively, indicating no difference between those cells. in contrast, only minimal viral productions of about . tcid /ml (prrsv) and . × pfu/ml (pedv) were observed in cells maintained at • c, which was likely due to inefficient removal of the bound virus. these results indicated that ribavirin has no inhibitory effect on the virus entry process. like all positive-sense rna viruses, following the uncoating process, the nidovirus genome is released into the cytoplasm and immediately serves as a template for viral translation by the same cellular cap-dependent mechanism. early nidovirus translation produces replicase polyproteins that are posttranslationally cleaved into nsps by viral proteases. subsequently, the nonstructural replicase proteins mediate de novo synthesis of viral rna, including genomic rna replication and sg mrna transcription. the nidovirus structural proteins are translated lately from respective sg mrna transcripts. thus, to determine the inhibitory mechanism of ribavirin on the postentry steps of the nidovirus life cycle, we first investigated whether viral protein translation was affected by ribavirin. to accomplish this, pam-pcd and vero cells were treated with ribavirin for h prior to infection, and the drug was allowed to remain during infection and subsequent incubation. the expression levels of the prrsv n and pedv s proteins in the presence or absence of ribavirin were evaluated at hpi by western blot analysis. the production of both proteins productions was drastically diminished during ribavirin treatment (fig. ) . densitometric analysis of the western blots revealed that the intracellular expression of both n and s proteins was reduced by ribavirin, with a maximum of about % inhibition at the concentration of m and m, respectively (fig. ) . this marked reduction was probably not the result of a nonspecific decrease in the translation mechanism, since ponceau s staining results indirectly indicated that the expression levels of the overall cellular proteins remained similar following treatment (data not shown). taken together, these results demonstrated the inhibitory effects of ribavirin specifically against viral translation during porcine nidovirus replication. for positive-strand rna viruses, synthesis of the viral nonstructural proteins is required for viral rna replication and transcription. thus, it is conceivable that impaired viral protein expression in would be caused by inhibition of viral rna synthesis. since nidovirus infection produces two rna entities including genomic versus subgenomic, we therefore determined if ribavirin specifically affected genome replication and sg mrna transcription. to answer this question, the relative levels of both genomic rna and sg mrna were assessed by quantitative real-time strand-specific rt-pcr in the presence or absence of ribavirin upon porcine nidovirus infection. as shown in fig. a , ribavirin exhibited a maximal % and % reduction in the synthesis of prrsv genomic rna and sg after washing with cold pbs, infected cells were maintained in the presence or absence of ribavirin either at • c or • c for an additional hour. bound but uninternalized virus particles were removed by treatment with protease k. the infected cells were then serially diluted and plated onto fresh target cells. at days post-incubation, internalized viruses were titrated by ifa and plaque assay for prrsv (left) and pedv (right), respectively. results are expressed as the mean values from triplicate wells and error bars represent standard deviations. *p = . - . ; † p < . . mrna at a concentration of m, respectively, when compared with untreated infected cells. furthermore, a similar inhibitory effect of ribavirin on genome replication and sg mrna transcription of pedv was observed. the amounts of pedv genomic rna and sg mrna detected in cells treated with m ribavirin were only about % and % of the untreated level (fig. b) . the decreases in viral rna levels after the addition of ribavirin were not due to nonspecific inhibition of transcription, as mrna levels of the internal control (␤-actin or gapdh) remained unchanged in all samples (data not shown). altogether, these results indicated that ribavirin suppresses the synthesis of the nidoviral genomic rna and sg mrna. several mechanisms of action for the antiviral activity of ribavirin have been suggested, including a reduction in cellular gtp pools via inosine monophosphate dehydrogenase (impdh) inhibition and increased mutation frequency on the virus genome leading to error catastrophe (graci and cameron, ) . to assess these known mechanisms, we first examined whether replenishment of guanosine affected the antiviral effect of ribavirin against porcine nidovirus infection. the addition of m guanosine to the ribavirin-treated and virus-infected cells was found to moderately reverse the antiviral activity of ribavirin. the incubation of ribavirin alone reduced prrsv production to % and % at m and m, respectively, whereas supplementation of guanosine to ribavirin inhibited virus production to % and % at the same concentrations (fig. a, left panel) . likewise, while pedv production was declined to %, %, and % in the presence of ribavirin alone at m, m, and m, respectively, it decreased to %, %, and % at the same concentrations when guanosine was added to the ribavirin treatment (fig. a, right panel) . to verify these results, we also tested the effects of mpa, a potent inhibitor of cellular impdh, on the replication of prrsv and pedv. as shown in fig. b , mpa efficiently reduced porcine nidovirus propagation fig. . inhibition of viral protein translation by ribavirin. ribavirin-treated pam-pcd and vero cells were mock-infected or infected with prrsv (a) or pedv (b) for h and were further cultivated in the presence or absence of ribavirin. at hpi, cellular lysates were collected, resolved by sds-page, transferred to a nitrocellulose membrane, and immunoblotted by using the antibody that recognizes the prrsv n protein or the pedv s protein. the blot was also reacted with mouse mab against ␤-actin to verify equal protein loading. each viral protein expression was quantitatively analyzed by densitometry in terms of the relative density value to the ␤-actin gene and ribavirintreated sample results were compared to vehicle-control results. values are representative of the mean from three independent experiments and error bars denote standard deviations. *p = . - . ; † p < . . inhibition of viral rna transcription by ribavirin. pam-pcd and vero cells pretreated with ribavirin were mock-infected or infected with prrsv (a) or pedv (b) for h and were incubated in the presence of ribavirin. total cellular rna was extracted at hpi, and strand-specific viral genomic rnas (black bars) and sg mrnas (white bars) of prrsv and pedv were amplified by quantitative real-time rt-pcr. viral positive-sense genomic rna and sg mrna were normalized to mrna for porcine ␤-actin or monkey gapdh and relative quantities (rq) of mrna accumulation were evaluated. ribavirin-treated sample results were compared with untreated results. values are representative of the mean from three independent experiments and error bars denote standard deviations. *p = . - . ; † p < . . at concentrations greater than . m and . m in a dosedependent manner for prrsv and pedv, respectively. sequence analysis of the porcine nidovirus genome in the presence or absence of ribavirin was conducted to investigate whether it triggers catastrophic mutations. we analyzed recombinant sequences of the orf to orf -coding region corresponding to nucleotide numbers , - , (prrsv) and the n gene region corresponding to nucleotide numbers , - , (pedv) from each virus grown in the presence of ribavirin. as controls, colonies were also obtained individually from untreated prrsv-and pedv-infected a the prrsv orf -orf coding region ( bp) and the pedv n ( bp) gene were rt-pcr amplified and the amplicon product was then cloned into the pgem-t vector. for sequencing of the gene in the recombinant vector, clones per group were selected and analyzed the number (rate) of mutations. b a total of , nt and , nt for prrsv and pedv were sequenced and a mutation frequency was calculated per nt, respectively. cells. in the ribavirin-treated groups, point mutations occurred in ( %) plasmids for both prrsv and pedv, which corresponded to a mutation frequency of . and . per nt, respectively. interestingly, mutations were also found in ( %) plasmids in the untreated groups for prrsv and pedv, which were calculated as a mutation frequency of . and . per nt, respectively (table ) . these sequencing results indicated that there were no significant differences in the mutation rates of ribavirin-treated and non-treated groups upon porcine nidovirus infection. the nidovirales are an order of enveloped, positive-strand rna viruses with animal hosts, which synthesize a nested set of multiple sg mrnas. this order includes three families arteriviridae, coronaviridae, and roniviridae, which have similar genome organization and replication strategy, but different virion morphology and genome length (mayo, ; lai et al., ; snijder and spaan, ) . porcine nidoviruses are not only devastating viral pathogens to the pig population, but can also be applied as an animal virus model to study human or veterinary-important nidoviruses. among these, despite tremendous efforts to control the diseases, prrsv and pedv have continuously plagued pigproducing countries, leading to significant economic impacts on the global or asian swine industry, respectively. this is partially due to a lack of efficient vaccines capable of conferring full protection against viral infections and antiviral agents for treating porcine nidoviruses. although ribavirin has an antiviral effect on a variety of dna and rna viruses (sidwell et al., ) , its efficacy and mode of action on porcine nidovirus replication are currently unknown. the present study demonstrated that ribavirin also exerts very effective antiviral activity against prrsv and pedv in vitro, as indicated by ed values of approximately m ( . g/ml) and m ( . g/ml), respectively. ribavirin can potentially act via inhibition of various steps of the virus life cycle, including viral translation, genome or transcript capping, rna synthesis, and progeny virus spread (graci and cameron, ) . treatment of cells with ribavirin resulted in significant attenuation of postentry steps during the replication of porcine nidovirus, as determined by lower progeny production, diminished viral protein expression, and decreased synthesis of genomic rna and sg mrna. it was previously reported that the tier of murine norovirus (mnv- ) in the presence of ribavirin dropped about -fold after virus infection, but remained similar after h of virus infection (chang and george, ) . in this study, growth kinetics experiments also indicated that the reduced rate of nidoviral titers in the presence of ribavirin was found to be comparable between and hpi. since ribavirin should be converted to its monophosphate active form (rmp) to exert antiviral activity, it appears that virus infection may disrupt the normal metabolic processes, leading to interference with the conversion of ribavirin (parker, ) . taken together, our data indicate that ribavirin potently elicits antiviral activity against prrsv and pedv in target cells. an important aspect of the antiviral activity of ribavirin may be attributed to the ability to act via multiple mechanisms simultaneously. although the mechanisms of action of ribavirin still remain controversial, five primary mechanisms have been proposed depending on the particular virus. these include reduction in cellular gtp pools by inhibiting impdh, enhanced mutation frequency via its incorporation into the viral genome leading to catastrophic error, modulation of host immune responses, inhibition of mrna capping, and interference with viral rna polymerase. therefore, the mechanisms of action of ribavirin may differ among viruses, and its antiviral activity may be operated predominantly via one or two mechanisms (graci and cameron, ; parker, ) . to elucidate potential mechanisms responsible for the antiviral effect of ribavirin on porcine nidoviruses, we first tested whether the antinidoviral activity of ribavirin is involved in inhibition of impdh and subsequent depression of cellular gtp levels. in previous studies, the addition of guanosine to the culture medium efficiently reversed the antiviral effects of ribavirin against norovirus, yellow fever virus, and human parainfluenza virus (chang and george, ; leyssen et al., ) . the present study so investigated the effects replenishing gtp by adding guanosine to ribavirin-treated cells upon virus infection. consistent with previous reports, the addition of guanosine to the ribavirin treatment significantly reversed the antiviral activity of ribavirin in porcine nidoviruses. furthermore, a noncompetitive impdh inhibitor, mpa, was found to strongly inhibit the replication of prrsv and pedv at concentrations above . m and . m, respectively. in contrast to ribavirin, which has to be converted to the active rmp form to elicit the antiviral activity, mpa does not require metabolic activation to exert its function. thus, the efficient antinidoviral activity of mpa suggests that ribavirin may directly lead to the inhibition of intracellular impdh levels. since rna viruses replicate with high genetic variation, they exist as viral quasispecies that are complex and dynamic mutant distributions that share a dominant nucleotide sequence and a mutant spectrum (ruiz-jarabo et al., ) . therefore, rna viruses reside on the edge of mutation crisis, and the increased average error frequency can cause defective genetic information and reduced viability termed error catastrophe (crotty et al., ; day et al., ) . ribavirin has been shown to trigger catastrophic mutations including lethal mutations in a variety of viruses and these may accumulate as a virus goes through multiple rounds of replication, resulting in extinction of the viral population (crotty et al., ; contreras et al., ; day et al., ; lanford et al., ; severson et al., ) . however, sequence analysis of the conserved n gene regions of prrsv and pedv in the ribavirintreated or untreated groups revealed that the two groups produced similar mutation frequencies ( . / . versus . / . ). these results were not surprising since rna genomes mutate at average rates of − - − per nucleotide copied during replication of rna viruses by the virus-encoded rna-dependent rna polymerase (rdrp) lacking proofreading functions (drake and holland, ) . our data indicated that the antiviral activity of ribavirin against porcine nidoviruses may not be associated with error-prone replication by increasing the probability of ribavirin incorporation into the viral genome. in conclusion, our findings described here demonstrated that ribavirin effectively prevents the replication of porcine nidoviruses in target cells at subcytotoxic doses. additionally, the highest doses of ribavirin used in this study did not increase the frequency of mutations in the nidoviral genome, and instead affected 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and the effectiveness of ribavirin treatment memory in viral quasispecies porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-␤ production by inhibiting irf activation in immortalized porcine alveolar macrophages ribavirin causes error catastrophe during hantaan virus replication broad-spectrum antiviral activity of virazole: -beta-d-ribofuranosyl- , , -triazole- -carboxamide arteriviridae family coronaviridae proteolytic maturation of replicase polyprotein pp a by the nsp main proteinase is essential for equine arteritis virus replication and includes internal cleavage of nsp synergism between porcine reproductive and respiratory syndrome virus (prrsv) and salmonella choleraesuis in swine duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus virus-encoded proteinases and proteolytic processing in the nidovirales this research was supported by basic science research program through the national research foundation of korea (nrf) funded by the ministry of education, science and technology ( - ). key: cord- -xorj tnz authors: kao, chi-fei; chiou, hue-ying; chang, yen-chen; hsueh, cheng-shun; jeng, chian-ren; tsai, pei-shiue; cheng, ivan-chen; pang, victor fei; chang, hui-wen title: the characterization of immunoprotection induced by a cdna clone derived from the attenuated taiwan porcine epidemic diarrhea virus pintung strain date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: xorj tnz the porcine epidemic diarrhea virus (pedv) poses a great threat to the global swine industries and the unreliable protection induced by the currently available vaccines remains a major challenge. we previously generated a genogroup b (g b) pedv taiwan pintung (pedvpt) strain, pedvpt-p , and determined its promising host immune response against the virulent pedvpt-p strain. to study the attenuation determinants of pedvpt-p and establish a pedvpt-p -based recombinant vector as a vaccine platform for further antigenicity modification, ipedvpt-p , a full-length cdna clone of pedvpt-p , was established. comparing to the parental pedvpt-p virus, the ipedvpt-p virus showed efficient replication kinetics with a delayed decline of viral load and similar but much more uniform plaque sizes in vero cells. in the -week-old piglet model, fecal viral shedding was observed in the pedvpt-p -inoculated piglets, whereas those inoculated with ipedvpt-p showed neither detectable fecal viral shedding nor pedv-associated clinical signs. moreover, inoculation with ipedvpt-p elicited comparable levels of anti-pedv specific plasma igg and fecal/salivary iga, neutralizing antibody titers, and similar but less effective immunoprotection against the virulent pedvpt-p challenge compared to the parental pedvpt-p . in the present study, an infectious cdna clone of an attenuated g b pedv strain was successfully generated for the first time, and the in vitro and in vivo data indicate that ipedvpt-p is further attenuated but remains immunogenic compared to its parental pedvpt-p viral stock. the successful development of the ipedvpt-p cdna clone could allow for the manipulation of the viral genome to study viral pathogenesis and facilitate the rapid development of effective vaccines. the porcine epidemic diarrhea virus (pedv) is an enveloped, positive-sense and single-stranded rna virus, belonging to the order nidovirales, family coronaviridae, and genus alphacoronavirus [ ] . the genome of pedv is about kilobase pairs in length and comprises of seven open reading frames (orf), including orf a and b genes that constitute the two thirds of the genome and encode the replication complex; the spike (s) gene that governs viral entry; the envelop (e), membrane (m), and nucleocapsid (n) genes that are responsible for virion assembly; and the accessory orf gene with an undetermined function [ ] . the porcine epidemic diarrhea virus is the causative agent of porcine epidemic diarrhea (ped), a historic, highly contagious enteric swine disease characterized by diarrhea, dehydration, and the growth retardation in pigs of all ages [ ] . in late , new and highly virulent pedv strains arose in china and spread rapidly worldwide by late , resulting in nearly % mortality in the affected nursing piglets [ ] [ ] [ ] . to date, there are still indelible endemics and considerable economic losses in the global swine market [ ] . besides, the protection conferred by currently available vaccines is, unfortunately, unsatisfactory [ , ] . based on the nucleotide identity of the s gene, pedvs are categorized into four genogroups (gs), namely g a, g b, g a, and g b [ ] . among these, the g b pedv strains that predominate the field in asia and north america, show higher pathogenicity [ ] and appear to elicit broader protection across different genogroups [ ] [ ] [ ] . although the increased virulence of new pedv strains was ascribed to several mutations in the s gene through viral escape from antibody neutralization induced by traditional vaccines [ , ] , the detailed mechanism remains elusive. previously, we generated an attenuated g b taiwan pedv strain, pedv pintung passage (pedvpt-p ) virus, by serial passage of the parental pedvpt strain in vero cells [ ] . despite the high potential of pedvpt-p as a future vaccine candidate against pedv as indicated by its reduced pathogenicity and robust host immune response in our -week-old piglet model [ ] , the difficulty in pedv isolation and subsequent lengthy passage process rendered the pedvpt-p unable to promptly respond to the vast outbreak in late . this year, zhou et al. [ ] reported a new disastrous swine disease outbreak in china in caused by an hku -related coronavirus of bat origin, again highlighting the potential burden of the interspecies jumping of coronavirus, and a pressing need for a readily applicable vaccine platform for new emergences. the reverse genetics system has been widely used to study viral pathogenesis and novel vaccine design. at present, the reported pedv infectious cdna clones were exclusively constructed based on sequences of the representative wild-type pedv isolates [ ] [ ] [ ] [ ] [ ] . consequently, they are highly pathogenic and fatal in suckling piglets, comparable to their parental viruses. with regard to vaccine use, further genetic editing is necessary to attenuate these recombinant viruses. alternatively, a complementary approach exploiting the attenuated phenotype to address the safety concerns has not been described. in the present study, a full-length cdna clone of the attenuated pedvpt-p , ipedvpt-p , was generated. in addition, the pathogenicity, immunogenicity, and protection against virulent pedvpt-p challenge by ipedvpt-p were evaluated in the -week-old piglet model. our data suggest that the ipedvpt-p virus was more attenuated but able to elicit similar immunogenicity and immunoprotection against the autologous virulent pedvpt-p challenge compared to the parental pedvpt-p virus. this ipedvpt-p cdna clone is expected to allow for the manipulation of the viral genome to study viral pathogenesis. on the other hand, it can serve as a vaccine platform, for example, by directly replacing the s gene with that of the (re)emerging swine coronaviruses. vero c cells (atcc no. crl- ) were maintained in dulbecco's modified eagle's medium (dmem, gibco, grand island, ny, usa) supplemented with % fetal bovine protein (fbs), ng/ml amphotericin b, u/ml penicillin, and µg/ml streptomycin. viral stock of pedvpt-p in post-inoculation medium (pi medium) containing dmem supplemented with . % tryptose phosphate broth (tbp), . % yeast extract ( . %), and µg/ml trypsin, as prepared in our previous study [ ] , was used for generating the infectious cdna clone and as the control for in vivo and in vitro studies, whereas the virulent pedvpt-p virus was used for animal challenge. the strategy used to engineer the full-length cdna clone of pedvpt-p , namely ipedvpt-p , was modified according to a previously published method [ ] . briefly, the complete genome of pedvpt-p (genbank accession no. ky ) was divided into six fragments by pcr amplification using primer pairs (table ) incorporated with specific type-iis restriction enzyme sites for seamless ligation. fragment a contained a t promoter sequence at its end to allow in vitro transcription and the sequence was designed following the article published previously [ ] ; a -adenosine sequence was added to the end of fragment f to simulate polyadenylation. a naturally occurring bsai site in fragment e was removed by introducing a silent mutation (c t) by site-directed mutagenesis according to the previously described protocol [ ] . an additional synonymous mutation (t c) was generated near the junction of fragments e and f, to create a novel bsmbi recognition site. pcr amplicons of all fragments were subcloned into plasmid vectors (pjet . ; thermo fisher scientific, waltham, ma, usa). each subclone was digested with the corresponding type-iis restriction enzymes as indicated in figure and gel-purified using a qiaquick gel extraction kit (qiagen, hilden, germany). assembly of the full-length cdna was conducted by employing t ligase (neb, ipswich, ma, usa) overnight at • c. the ligated full-length cdna was phenol-chloroform extracted and in vitro transcribed to full-length rna transcripts using a mmessage mmachine t transcription kit (ambion, austin, ca, usa) following the manufacturer's instructions. the cap analog to gtp ratio was adjusted to : to increase the yield of full-length transcripts. to facilitate viral recovery, nucleocapsid (n) transcripts were also generated from amplicons flanking the entire n gene with the addition of the t promoter sequence and poly-a tail at the and ends, respectively. the n transcripts were precipitated using lithium chloride (ambion, austin, ca, usa) and purified with ethanol. the reaction mixture of full-length transcripts ( µl) and µg of n transcripts were mixed thoroughly and electroporated into vero cells in a suspension of µl with cells/ml in rnase-free phosphate buffered saline (pbs) using a gene pulser xcell™ electroporation system (bio-rad, hercules, ca, usa), with four pulses at v, µf and - s rest between each pulse. after electroporation, the cells were initially incubated at room temperature for min and then resuspended in dmem supplemented with % fbs in a -cm flask overnight at • c to allow for recovery. on the next day, the cells were washed twice with dulbecco's phosphate-buffered saline (dpbs) and maintained in pi medium for an additional - days until cytopathic effects (cpe) characterized by cell fusion, syncytial cell formation, and cell detachment were observed. the whole flask was subjected to one freeze-and-thaw cycle and the rescued virus was passaged once to generate a viral stock of ipedvpt-p for further use. the viral stock was titrated in vero cells in a -well plate to determine viral titer. to detect the pedv antigen, immunocytochemistry (icc) was performed as described previously [ ] . briefly, vero cells infected with ipedvpt-p showing typical cpe were fixed with % ice-cold acetone, air-dried, and incubated with an in-house anti-pedv n antibody at room temperature (rt) for h. after washing three times with pbs, a polyclonal anti-rabbit/mouse immunoglobulin, envision-dab + system (dako, carpinteria, ca, usa), was applied for h at rt. following three washes with pbs, the cells were incubated with , -diaminobenzidine (dab) chromogen from a peroxidase dab substrate kit (dako, carpinteria, ca, usa) according to the manufacturer's instructions. positive signals were visualized under an inverted light microscope (nikon, tokyo, japan). sequence analysis was conducted as described previously [ ] and a primer pair (sf and n- listed in table ) targeting the genome between nucleotides to was used to identify the presence of marker mutations, c t and t c, in the ipedvpt-p viral stock. the growth characteristics of ipedvpt-p and pedvpt-p in vero cells were evaluated and compared by examining the growth kinetics and plaque morphologies. confluent monolayers of vero cells were prepared on -well plates and infected with each virus at the multiplicity of infection (moi) . for h at • c in triplicate. to determine the growth kinetics, cells were washed twice with dpbs and then maintained in the pi medium. the supernatants at indicated time points, , , , and h post-inoculation (hpi), were collected and subjected to titration in vero cells seeded in -well plates. plaque assays were performed to characterize the plaque morphologies. after the adsorption of pedvs at moi . , vero cells were washed twice with dpbs and overlaid with pi medium containing % agarose (invitrogen, carlsbad, usa) pre-warmed to • c. upon solidification of the overlays, the plates were incubated for days at • c to allow pedv-infected vero cells to produce distinct plaques. the cells were fixed in . % neutral formalin for h at rt. the semisolid overlays were then removed manually and the cells were stained with % crystal violet in % ethanol and distilled water for min. the viral plaques were inspected after washing off the crystal violet, rinsing the plates with water, and air-drying at rt. the diameters of representative plaques for each virus were measured and compared. fifteen, -week-old, large white × duroc, crossbred piglets that were pedv-seronegative and pedv-shedding negative were selected from a conventional pig farm with no history of a g b pedv strain infection. these piglets were randomly assigned to three groups, including the pedvpt-p group (n = ), ipedvpt-p group (n = ), and mock group (n = ), and acclimated for one week prior to inoculation. at weeks of age, the piglets in each group were orally inoculated with ml of × tcid /ml of pedvpt-p , × tcid /ml of the pedvpt-p virus, or pi medium, respectively. to evaluate the protective efficacy induced by ipedvpt-p , piglets at weeks of age in all groups were orally challenged with ml tcid /ml of pedvpt-p . the animal experimental procedure was reviewed and approved by the institutional animal care and use committee (iacuc) of the national taiwan university (taiwan, republic of china) with approval no.: ntu el- . fecal consistency was monitored daily and scored visually as = normal, = loose, = semi-fluid, and = watery, as described previously [ ] . the body weight of each piglet was measured weekly. to quantitate the viral rna in stools, fecal samples collected from rectal swabs were resuspended in µl dpbs, pulse-vortexed for s and precipitated by centrifugation at , rpm for min. rna was extracted automatically from µl of stool suspension on a qiacube using the cador pathogen qiacube ht kit (qiagen, hilden, germany) according to the manufacturer's instructions. cdna was reverse-transcribed using a quantinova reverse transcription kit (qiagen, hilden, germany) following the manufacturer's protocols and it was used for quantitative real-time pcr analysis (qpcr). qpcr was conducted using the previously published primer-probe set [ ] and a quantinova ® probe pcr kit (qiagen, hilden, germany) on a cfx thermal cycler (bio-rad, hercules, ca, usa). the thermal profile comprised an initial denaturation at • c for min followed by cycles of • c for s followed by • c for s. the detection limit of the assay was determined by generating standard curves from serial -fold dilutions of known amounts of in vitro transcribed rna followed by reverse transcription and qpcr quantification as described above. the detection limit was calculated as rna copies per ml. to detect pedv specific plasma igg and fecal and salivary iga, an in-house, pedv s protein based indirect enzyme-linked immunosorbent assay (elisa) was conducted as described in the previous study [ ] . in brief, -well, flat-bottom microtiter plates (nunc, roskilde, denmark) were coated with µg/ml purified recombinant pedvpt s protein ( ng/well) and incubated overnight at • c. the plates were washed six times with µl of pbst (pbs containing . % tween ) and then blocked with µl of blocking buffer ( % bovine serum albumin in pbs) at rt for h. for the detection of plasma igg, µl of -fold diluted plasma samples in blocking buffer were added following six washes and incubated at rt for h. for fecal and salivary iga, µl of eluted fecal suspension and saliva at : dilution in blocking buffer were added following six washes and kept overnight at • c. after incubation, the samples were discarded and the plates were washed six times. to detect plasma igg, and the fecal and salivary iga, µl of either horseradish peroxidase (hrp) conjugated goat anti-pig igg (kirkegaard & perry laboratories, milford, ma, usa) at : dilution, or goat-anti-pig iga (abcam, cambridge, uk) diluted : , were added, respectively, and incubated at rt for h. following a wash step, µl of tetramethylbenzidine (tmb) substrate solution (kirkegaard and perry laboratories) was added to allow color development at rt for min. the reactions were terminated by adding µl of tmb stop solution (kirkegaard and perry laboratories) to each well. the optical density (od) at nm was measured on an elisa reader (molecular devices, sunnyvale, ca, usa). the antibody titers were expressed as sample-to-positive control ratio (s/p ratio) values. plasma samples of piglets were heated at • c for min to inactivate complement prior to use. for each well, mixtures containing µl of pedvpt-p virus ( viral particles) and µl of -fold diluted plasma samples in pi medium were incubated at • c for h before applying to vero cells ( × /well). after incubation with the virus-plasma mixture for h, vero cells were washed twice and maintained in pi medium for h. cytopathic effects were detected using inverted light microscopy (nikon, tokyo, japan). the neutralizing titer was defined as the highest dilution without cpe. the results of body weight, antibody titers, viral titer of growth kinetics at each time point, and fecal viral shedding were analyzed statistically on graphpad prism . (graphpad software, san diego, ca, usa) with two-way anova by time. a p value less than . was considered statistically significant. to generate the full-length cdna clone of ipedvpt-p , the complete genome sequence of pedvpt-p was split into six fragments by designing primer pairs fused with specific type-iis restriction enzymes. after the restriction enzyme digestion and dna ligation, transcripts containing the full-length ipedvpt-p sequence were successfully prepared. typical pedv-associated cytopathic effects in vero cells, characterized by multinucleated giant syncytia, were observed at about one day post-electroporation ( figure a) . the presence of ipedvpt-p was confirmed by the detection of the pedv n protein by immunocytochemistry ( figure a ) and sequence analyses for identification of the introduced substitutions, c t and t c ( figure b ). after an additional day, recombinant viral supernatants were harvested when the cpe reached % of the confluent monolayer. an ipedvpt-p viral stock with a titer of . × tcid /ml was prepared by an additional passage of the viral supernatants in the vero cells. to characterize the recombinant ipedvpt-p , plaque morphologies and growth kinetics of the recombinant ipedvpt-p in vero cells were compared to those of the parental pedvpt-p virus. interestingly, aside from the constantly short diameter, the plaque size of ipedvpt-p appeared to be more uniform than that of pedvpt-p ( figure c ). as to the replicative capacity, the multistep growth kinetics of both the ipedvpt-p and pedvpt-p viruses in vero cells were examined. at moi of . , while pedvpt-p replicated rapidly to the titer of . ± . tcid /ml at hpi, reached the peak viral titer of . ± . tcid /ml at hpi, and showed a declined viral titer of . ± . tcid /ml at hpi. ipedvpt-p showed a steady increase in viral progenies and a significantly decreased viral titer of . ± . tcid /ml at hpi, a comparable viral titer of . ± . tcid /ml at hpi, and a significantly high viral titer of . ± . tcid /ml at hpi ( figure d ). at mois of . and . , similar to those observed at . moi, ipedvpt-p replicated slower in the beginning, reached a similar peak viral titer as compared to pedvpt-p and showed a relatively slower reduction of viral titer after reaching the peak titer (see figure s ). to evaluate the pathogenicity of the ipedvpt-p virus, -week-old crossbred piglets were orally inoculated with ml of × tcid /ml of the ipedvpt-p virus, pedvpt-p virus, or pi medium. the clinical impact of viral inoculation in each piglet was evaluated by daily clinical fecal scoring, fecal viral shedding, and weekly body weight changes. during the study, no significant difference in body weight was revealed among the different groups at any indicated time points (figure ). while one piglet in the pedvpt-p group showed intermittent loose diarrhea (score = ) and viral shedding at to days post-inoculation (dpi) with the peak viral titer of . ± . log rna copies/ml at dpi (figure ) , no evidence of pedv-associated clinical signs and fecal viral shedding were demonstrated in both ipedvpt-p and mock groups. after the oral inoculation of piglets with the ipedvpt-p and pedvpt-p viruses, systemic pedv-specific igg in blood and mucosal specific iga in feces and saliva were measured at the indicated time points. compared to the mock group, an elevated mean s/p ratio of blood pedv-specific igg was detected at dpi and sustained until dpi in piglets from both the ipedvpt-p and pedvpt-p groups ( figure ) . moreover, subtle increases in the mean s/p ratios of fecal and salivary pedv-specific iga were also observed in both the ipedvpt-p and pedvpt-p groups compared to those of the mock group at dpi ( figure ). after oral inoculation with the virulent pedvpt-p virus, the daily fecal viral sheddings and fecal consistencies in all groups were evaluated. similar to our previous data [ ] , all piglets in the mock group showed the differing severity of clinical signs for diarrhea at - days post-challenge (dpc) (figure ) . the mean value of the pedv rna copies in feces of the mock group became detectable at dpc, shot up to a peak titer of . ± . log rna copies/ml at dpc, and then declined to a constantly low viral load of approximately log rna copies/ml at - dpc. in the pedvpt-p group, two piglets showed loose diarrhea (score = ) for one day at and dpc, respectively. the onset of fecal viral shedding in the pedvpt-p group after the challenge was further delayed to dpc compared to the other two groups. the highest viral titer of the pedvpt-p group also appeared at dpc, calculated as . ± . log rna copies/ml. in the ipedvpt-p group, all pigs remained clinically normal without an observation of diarrhea. compared to the mock group, only three of the five pigs in the ipedvpt-p group established fecal viral shedding through the experiment, with a delayed onset and a steadily fluctuated low viral shedding, ranging from the highest titer of . ± . to log rna copies/ml to undetectable levels ( figure ). in the mock group, the mean value of the s/p ratio of the systemic pedv-specific igg titers in the blood of piglets remained unchanged prior to the pedvpt-p challenge, after which the mean s/p value rose to . ± . at dpc ( figure ). on the other hand, the mean values of systemic pedv-specific igg in blood from the ipedvpt-p and pedvpt-p groups increased sharply to . ± . and . ± . at dpc, respectively, which was significantly higher than that of the mock group ( figure ). as for mucosal immunity, similar to the trend noticed in the systemic pedv-specific igg in blood, significant increases in the mean s/p values of both fecal and salivary anti-pedv specific iga were noted in the ipedvpt-p and pedvpt-p groups after challenge ( figure ). among them, the s/p values of salivary anti-pedv specific iga, . ± . and . ± . , in the ipedvpt-p and pedvpt-p groups, respectively, were significantly higher than those of the mock group ( . ± . ) at dpc (figure ) . the vn antibody titers of piglets in each group are depicted in figure . similar to the trend observed in systemic plasma igg titers, the ipedvpt-p group showed a comparable vn antibody titer against pedvpt-p to that of the pedvpt-p group through the study. the mean vn antibody titers of both groups increased mildly at dpc and spectacularly at dpc and were higher than those of the mock group during the study (figure ). data were expressed as the mean ± standard deviation. statistically significant differences between each group were examined using a two-way anova by time. in the present study, we described the first development of an infectious cdna clone of ipedvpt-p , and evaluated it's in vitro and in vivo characteristics. compared to the parental pedvpt-p virus, ipedvpt-p replicated more slowly in the beginning and reached a similar peak viral titer with similar but more uniform plaque sizes, suggesting that the composition of viral quasispecies in ipedvpt-p is less complex than that in the original pedvpt-p stock. moreover, neither fecal pedv rna shedding nor a pedv-associated clinical illness was detected in conventional -week-old piglets inoculated with the ipedvpt-p virus, indicating a further attenuated phenotype in vivo. importantly, piglets in the ipedvpt-p -inoculated group showed comparable levels of anti-pedv specific plasma igg, fecal/salivary iga, plasma neutralizing antibody titers, and a weakened but modest immunoprotection against the virulent pedvpt-p challenge compared to the parental pedvpt-p -inoculated piglets. taken together, our results suggest that ipedvpt-p is immunogenic in piglets and could be a potential safe viral vector candidate for vaccine development. while inoculation with ipedvpt-p was demonstrated to completely protect the piglets from developing diarrhea after challenge with the virulent pedvpt-p , the ipedvpt-p -inoculated group showed an earlier onset and longer duration of fecal pedv rna shedding with a higher peak value than that of the parental pedvpt-p -inoculated group. these data suggested that ipedvpt-p conferred a relatively weakened protection than that of the parental pedvpt-p . considering that the major variation between the pedvpt-p and ipedvpt-p viruses should be the heterogeneity of viral population as noted in plaque assays wherein the ipedvpt-p virus produced more uniform plaques than those of the parental pedvpt-p virus in vero cells, we speculate that the decrease in quasispecies diversity in ipedvpt-p may partly contribute to its further attenuation in vivo. to investigate the speculation, we conducted the next generation sequencing (ngs) to determine the quasispecies diversity of both viruses by exploring the recovered variants against our previous published sequence of pedvpt-p generated by sanger sequence (see table s ). the results clearly demonstrated that the pedvpt-p carried a greater sequence diversity than that of the ipedvpt-p , including single nucleotide variants (snv) and resultant amino acid substitutions; of interest, seven snv were found in the spike gene and of them were the same as the virulent pedvpt-p . for ipedvpt-p , excluding the artificially introduced marker mutations and those derived from the snv of pedvpt-p upon the initial construction of subclones of ipedvpt-p , only snv were uncovered. indeed, generating viruses with high-fidelity replication has been proposed as a rational strategy to develop genetically stable and safe attenuated vaccines [ , ] . however, for attenuated viruses, the in vivo fitness and antigenicity are already abated after serial cell culture passage. on the basis of quasispecies theory that the cooperative interplay between different variants determines viral characteristics including virulence [ , ] , it is possible that the consensus sequence used for constructing ipedvpt-p no longer sustained the original affinity of virus-host interaction or viral replication in enterocytes, which therefore impaired viral entry and/or limited the viral infection. furthermore, the limited diversity of the s gene in the ipedvpt-p viral stock might also diminish the potency and protective broadness of the induced antibody responses explaining the comparable level of systemic and mucosal antibody response but weakened protection in the ipedvpt-p -inoculated group. this speculation could be tested by comparing the tissue tropism and the quantity of pedv antigens of both pedvpt-p and ipedvpt-p in enterocytes using a -day-old piglet model. other explanation for the attenuation of ipedvpt-p might be attributed to the possible addition of the five nucleotides "ggaga" at the very extreme of the utr based on our primer design (see table ). considering the location that these additional nucleotides sequence should neither alter the secondary structures of the sl stem-loop, that serve as cis-acting elements required for driving subgenomic rna synthesis nor change any consensus transcription regulatory sequences -xua(a/g)ac- , we assumed that the effect of these additional nucleotides in the rescued ipedvpt-p virus on replication might be minimal. nonetheless, further studies are required to determine the potential effect of these five additional nucleotides on ipedvpt-p replication. in the present study, the profile of fecal pedv rna shedding and the pattern of antibody responses after inoculation with pedvpt-p appeared milder than those observed in our previous findings despite the fact that the same viral stock was used and that the conventional piglets used in this study were purchased from the same pig farm and housed in the same animal facility as described previously [ ] . that is, it seemed that the conventional piglets used in the current study were more resistant to the pedvpt-p infection. several host factors such as genetic variation, type of feed, gut microflora, and immune status among different litters at the time of inoculation may play an important role in the varying severity of clinical outcomes and immune responses [ ] . ideally, the discrepancy between the previous experiment might be minimized by expanding the size of groups, particularly if we chose conventional piglets as our target animals. nevertheless, due to the difficulty in collecting large numbers of pedv-negative piglets in taiwan where ped has become endemic, we decided to use five piglets per treatment to reach a statistical effect practice. although the use of conventional pigs often magnifies those variables and results in higher variation in the experimental results, to mimic the pathogenicity and immunoprotection of pedv in field conditions, the conventional pig model is still a preferred as a preclinical trial model. since the sudden appearance of the severe acute respiratory syndrome-coronavirus (sars-cov) in the early st century, the emergence and re-emergence of coronaviruses continually threatens the global public and animal health by causing severe illnesses, by having a high potential of zoonosis, and by causing great economic losses, indicating a desperate need for an effective and readily responsive vaccine platform. the large genome size of coronaviruses warrants a great tolerance for foreign genes and subsequent expression of heterologous antigens [ , ] . in addition, the insertion of other antigens by replacing the accessory orf gene or creating a novel expression cassette is also an attractive approach to design multivalent vaccines [ , , ] . under this concept, ipedvpt-p could be a potential safe vaccine backbone that facilitates the prompt generation of chimeric vaccines with the induction of mucosal immunity. for instance, viral antigenicity can be manipulated by replacing the ipedvpt-p s gene with that of other pedv or emerging swine coronaviruses although the potential loss of attenuation due to the s substitution requires further clarification. however, it is noteworthy that the inherent genetic instability of coronaviruses due to high recombination and mutation frequency is also a matter of concern as it raises the possibility of virulence reversion and acquisition of new tropism [ ] . besides, the intrinsic characteristics of the gene of interest, the targeted locus within the coronavirus genome, may also affect the expression level and stability of the recombinant viruses [ ] . accordingly, further characterization of the virulence variation and genetic stability of ipedvpt-p after different genetic modifications needs to be conducted. in this article, we described the first successful construction of an attenuated g b pedv infectious cdna clone of the ipedvpt-p virus and demonstrated the maintenance of fitness in vitro along with further attenuation in vivo. we also proposed that the initial low quasispecies diversity and the additional nucleotides at the end of ipedvpt-p may contribute to a further attenuated phenotype and potentially less effective immunoprotection against the virulent strain challenge. based 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university for ngs data analysis. the authors declare no conflict of interest with respect to the research, authorship, and/or the publication of the article. key: cord- - vi m authors: mizutani, tetsuya title: signaling pathways of sars-cov in vitro and in vivo date: - - journal: molecular biology of the sars-coronavirus doi: . / - - - - _ sha: doc_id: cord_uid: vi m severe acute respiratory syndrome (sars) is a respiratory illness with variable symptoms that was recognized as the first near-pandemic infectious disease of the twenty-first century. a novel human coronavirus, named sars coronavirus (sars-cov), derived from sars patients was reported as the etiologic agent of sars. studying the signaling pathways of sars-infected cells is key to understanding the molecular mechanism of sars viral infection. cell death is observed in cultured vero e cells after sars-cov infection. from sars-cov infection to cell death, p mitogen-activated protein kinase (mapk) is a key participant in the determination of cell death and survival. two signaling pathways comprising signal transducer and activator of transcription (stat ) and p ribosomal s kinase (p rsk) are downstream of p mapk. akt and jnk (jun nh( )-terminal kinase) signaling pathways are important to establish persistent infection of sars-cov in vero e cells. expression studies of sars-cov proteins indicate that the viral proteins are able to activate signaling pathways of host cells. the study of signaling pathways in sars-cov patients is difficult to perform compared with in vitro studies due to the effects of the human immune system. this review highlights recent progress in characterizing signal transduction pathways in sars-cov-infected cells in vitro and in vivo. . sars was first recognized in china in november and subsequently spread to other countries, thus emerging as the first nearpandemic infectious disease of the twenty-first century. a worldwide total of , cases of sars, of which ( . %) resulted in death, was reported by the world health organization (who) (http://www.who.int/csr/sars/country/table _ _ /en/index.html). sars-cov belongs to the coronaviridae family (order nidovirales) of enveloped single-stranded positive rna viruses (marra et al. ; rota et al. ; thiel et al. ) . the sars-cov genome is approximately kb in length and is the longest known amongst the rna virus genomes. the sars-cov genomic rna has a cap structure and a poly-a tail at the and ends, respectively. sars-cov genome replication occurs in the cytoplasm. during viral replication, a full-length genomic negative-stranded rna is transcribed from genomic positivestranded rna by the viral rna polymerase that is initially translated from genomic rna. approximately % of sars-cov genomic rna encodes viral polymerase and its related proteins. the mrna transcription of coronavirus is unique, because all mrnas have a nested set structure. the mrnas have a leader sequence of approximately nucleotides and poly-a tails at the end. mouse hepatitis virus (mhv), which is a prototype of coronavirus, has seven mrnas, whereas sars-cov has at least nine mrnas. the leader rna is transcribed from the end of full-length genomic negative-stranded rna. there is strong evidence that the leader rna is transcribed as small sized-rna, which is approximately bases in length. the leader rna binds to intragenic initiation sites on negative-stranded rna, and then viral rna polymerase starts to transcribe mrna at the site. the sars-cov viral genomic rna comprises open reading frames (orfs), and eight of the encoded proteins are unique compared with other coronaviruses. these unique proteins are thought to be involved in the pathogenetic mechanism of sars-cov. large overlapping polyproteins ( a and b) encoded by approximately % of the sars-cov viral genome are processed into nonstructural proteins including polymerase and proteases (chymotrypsin-like cysteine protease and papain-like protease). these proteins are thought to be essential in viral replication and transcription. the viral particle of sars-cov mainly consists of four structural proteins, spike (s), membrane (m), envelope (e), and nucleocapsid (n) (fig. . ). the viral particle may also comprise viral accessory proteins that bind to the structural proteins. the s protein binds to the viral receptor of host cells and enables the virus to enter the cytoplasm by endocytosis. sars-cov has the potential to cause respiratory illness in human patients. cytokine storm occurs in sars-cov-infected patients and is one of the observed pathologic mechanisms of sars-cov infection. on the other hand, apoptotic cell death is observed in vitro when sars-cov-sensitive cultured cells such as vero e cells are used (mizutani et al. c) . various signaling pathways are activated during the entire process of viral infection, from s protein-ace (angiotensinconverting enzyme- ) binding for internalization into the host cells to apoptotic cell death (mizutani ) . the most common signaling pathways are mitogen-activated protein kinase (mapk) pathways, which include jun nh -terminal kinase (jnk), extracellular signal-regulated kinase (erk), and p mapk. these three major mapks are highly conserved in a wide range of species from yeast to mammals and are regulatory proteins of cell death and cell survival in living cells. thus, mapks are key to the process of apoptosis (garrington and johnson ) . mapkk kinase (mapkkk) activates mapk kinase (mapkk), and then mapkk activates mapk. generally, the erk signaling pathway promotes cell survival and proliferation, and jnk and p mapk induce apoptosis. however, the role of each mapk varies depending on cell type and stimulation. many signaling pathways are activated in virus-infected cells, and cross-talk activation between signaling pathways occurs. thus, signaling pathways regulating cell death and survival in virus-infected cells is highly complex. analysis of activated signaling pathways in sars-cov-infected cells and patients is required for understanding the pathogenesis of sars. this review highlights recent progress in characterizing signal transduction pathways induced by sars-cov infection in vivo and in vitro. the p map kinase is expressed in response to stressors, and viral infection generally induces activation of p mapk. the roles of p mapk in viral infection/replication have been researched recently as described below. environmental stresses, such as uv irradiation, oxidative stimuli and proinflammatory cytokines, are able to induce activation of p mapk. there are at least four isoforms of p mapk: p a, p b, p g, and p d _ ; platanias ; lee et al. ), but these isoforms are generally not distinguished in the field of virology. however, these four isoforms exhibit different properties and have mapks have more than % similarity at the amino acid sequence level, and their functions are inhibited by the pyridinyl imidazole inhibitor sb [ -( -fluorophenyl)- -( -methylsulfinylphenyl)- -( -pyridyl) imidazole]. conversely, p g and p d mapks, which have % similarity to p a, are not inhibited by sb . furthermore, p a and p b mapks are widely expressed in tissues, whereas the expression of p g and p d mapks is tissue-specific. in the field of virology, because sb is generally used as an inhibitor of the p mapk signaling pathway, it can be used in studying the role of p a and/or p b mapks in sars-cov infection. the kinases upstream of p mapk are mkk (mapk kinase ) and mkk (mapk kinase ), which are known to phosphorylate and activate p mapk. interestingly, mkk has affinity to pkr in the presence of double-stranded rna, poly(ri;rc) and pkr is able to activate mkk , but not mkk . this result indicated that interaction of mkk and pkr provides a mechanism to regulate activation of p mapk (silva et al. ) . in hepatitis c virus-core-expressing cells, pkr has an important role in cell cycle arrest and was shown to interact strongly with p mapk (spaziani et al. ) . as upstream of p mapk, transforming growth factor (tgf)-b-activating kinase (tak ), apoptosis signal-regulatory kinase (ask ), and mapkkk are known as mapkkks. downstream targets of p mapk are well-known as mitogen and stressactivated protein kinase (msk ), map kinase-interacting kinase (mnk ), and mapk-activated protein kinase and (mapkapk and ) (freshney et al. ). these signaling pathway proteins have further downstream targets. for example, mnk activates the eukaryotic translation initiation factor e (eif e) (gingras et al. ) . mapkapk activates heat shock protein (hsp ), camp response element-binding protein (creb), and transcription factor- (atf- ) (garrido et al. ; tan et al. ) . mouse hepatitis virus (mhv) a strain induces interleukin- (il- ) production via eif e phosphorylation under p mapk activation (banerjee et al. ) . inhibitors of p mapk inhibit transcription of viral mrna and production of viral protein, indicating that p mapk is utilized to promote viral protein synthesis. conversely, p mapk enhances transcription of chop (c/ebp homologous protein) encoded by the growth arrest-and dna damage-inducible gene (gadd ) (wang et al. ) . the p mapk-induced apoptosis via activation of the chop pathway occurs in cells infected with japanese encephalitis virus (jev) (su et al. ) . the h n subtype of influenza virus induces tumor necrosis factor alpha (tnf-a) expression via activation of p mapk (lee et al. ) . the p mapk signaling pathway is thought to primarily induce apoptosis in virus-infected cells. as described in hcv infection, p mapk is able to promote cell survival. although p mapk is activated in many cases of viral infection, viral proteins sometimes negatively regulate p mapk. the orf protein of varicella-zoster virus (vzv) is known to repress phosphorylation of p mapk for negative regulation of cellular proinflammatory responses (rahaus et al. ). thus, activation or inactivation of p mapk occurs in the pathogenesis of disease caused by viral infection. the p mapk signaling pathway takes part in cell death, as previously described. apoptosis is an active and physiologic type of cell death and is a host cell's protective mechanism for preventing the spread of viral particles before production of viral particles. vero and vero e cells, which are monkey kidney cells, are widely used in sars-cov research because of their high susceptibilities to infection due to lack of interferon genes. apoptosis has been shown to be inducible by infection with sars-cov (mizutani et al. c; yan et al. ) . cytopathic effects (cpes), defined as focal cell rounding and dna fragmentation typical of apoptosis, are observed in sars-cov-infected vero e cells at h post-infection (h.p.i.) (mizutani et al. c ). activated caspase , which has an essential role in apoptosis, was detected at peak levels at h.p.i. on the other hand, the phosphorylation level of p mapk reached a maximum at h.p.i. in virus-infected cells. the phosphorylated p mapk was active, as shown by using an in vitro kinase assay. the cpe observed in sars-cov-infected cells is slightly inhibited by sb , and therefore p mapk activation is thought to induce cpe of virus-infected cells. however, dna fragmentation is not inhibited by the inhibitor. apoptosis and cpe are thought to be linked, and activation of p mapk is a promoter of cell death in vero e cells infected by sars-cov. however, sb treatment of vero e cells indicates that there is no requirement for p mapk activation in sars-cov replication. the p mapk signaling pathway perhaps has other roles in sars-cov-infected cells. the downstream targets of p mapk are phosphorylated in sars-covinfected cells. the level of phosphorylated eif e is increased in sars-covinfected cells (mizutani et al. c ). however, the activated eif e does not regulate viral protein synthesis, as demonstrated by the similar kinetics of viral protein accumulation in infected vero e cells in the presence and absence of sb . both mapkapk- and its substrate hsp- are phosphorylated in virus-infected cells. hsp- is known as an anti-apoptotic protein as it inhibits apoptosome formation (garrido et al. ) . creb is also known to mediate a survival signal under various conditions (tan et al. ; ginty et al ; von knethen et al. ) , and creb is also phosphorylated in sars-cov-infected cells. the expression of sars-n protein in transfected cos- cells induces phosphorylation of p mapk, hsp- , and creb (surjit et al. ) , whereas the viral-n protein expression system of vaccinia virus (dis-n) does not induce phosphorylation of p mapk (mizutani et al. d ). activation of the p mapk pathway induces actin reorganization in cos- cells devoid of growth factors (surjit et al. ) . furthermore, the a protein of sars-cov induces apoptotic cell death and phosphorylation of p mapk in t cells (kopecky-bromberg et al. ). however, sb does not prevent cell rounding, apoptosis, and chromatin condensation induced by the a protein. the differences in the results are most likely due to the use of different cell cultures and expression systems. overall, phosphorylated proteins downstream of p mapk have the potential to induce an anti-apoptotic environment in sars-cov-infected cells. however, activated p mapk in sars-cov-infected cells is thought to be able to promote both cell death and survival. perhaps there are other substrates of p mapk that are inducible on cell death of vero e cells caused by sars-cov infection, or perhaps there is cross-talk between the p mapk signaling pathway and other signaling pathways inducing cell death. in vero e cells, signal transducer and activator of transcription (stat) protein is constitutively phosphorylated at tyr- and is slightly phosphorylated at ser- (mizutani et al. a ). sars-cov infection is able to induce dephosphorylation of stat tyr- after h.p.i. on the other hand, ser- -phosphorylated stat is slightly increased at the same point in time. the activity of stat transcription factors is induced by phosphorylation of a single tyrosine residue, leading to dimerization via an intermolecular sh phosphotyrosine interaction (shuai et al. (shuai et al. , schindler et al. a schindler et al. , b . stat is known to be activated in response to interleukin- (il- ) and il- , and is thought to act as an anti-apoptotic transcription factor (rajan and mckay ; grandis et al. ; mora et al. ) . tyr- phosphorylation of stat is necessary for its activation (shuai et al. (shuai et al. , schindler et al. b) , suggesting that sars-cov infection leads to a decrease in stat activation. furthermore, stat does not act as a transcriptional enhancer in sars-cov-infected vero e cells, as shown by the disappearance of tyr- -phosphorylated stat from the nuclear fraction post sars-cov infection. the proteins upstream of stat in the signaling pathway are janus kinases (jak and ) and tyk , which are phosphorylated at low levels in mock-infected vero e cells, even after virus infection. the signal transducing adaptor molecule (stam ), which is known to be associated with jak and jak via the immunoreceptor tyrosine-based activation motif, is upregulated in sars-cov-infected vero e cells (leong et al. ) . therefore, tyr- dephosphorylation of stat in virus-infected cells is independent of its upstream kinases, and there may be other signaling pathways regulating stat phosphorylation and dephosphorylation. two inhibitors of p mapk (sb and sb ) partially inhibit dephosphorylation of stat at tyr- , indicating that the p mapk signaling pathway is upstream of tyr- dephosphorylation of stat in sars-cov-infected vero e cells. inactivation of stat via p mapk activation may induce cell death in sars-cov-infected cells. however, the kinetics of stat after sars-cov infection varies for different cell types. the suppressors of cytokine signaling- (socs ) mrna are suppressed in sars-cov-infected caco- cells (okabayashi et al. ) , leading to continuous activation of stat . a serine/threonine kinase, p ribosomal s kinase (rsk), belongs to another signaling pathway, which is regulated by p mapk. generally, p rsk is phosphorylated at thr- by erk (gavin and nebreda ; smith et al. ) , and this phosphorylation induces autophosphorylation at ser- , and then pdk (phosphoinositide-dependent kinase ) phosphorylates at ser- (frödin et al. ; jensen et al. ; richards et al. ) . no significant differences are observed in phosphorylation levels of p rsk at ser- and thr- in sars-cov-infected vero e cells (mizutani et al. a ). however, ser- of p rsk is phosphorylated in virus-infected confluent cells. thus, phosphorylation of p rsk ser- is upregulated without upregulation of thr- in sars-covinfected cells. the phosphorylation of ser- is decreased in sb -treated virus-infected cells, indicating that p mapk can induce phosphorylation of ser- . furthermore, p rsk phosphorylates creb . in sars-cov-infected vero e cells, p mapk activation induces phosphorylation of p rsk ser- , and then creb is thought to be phosphorylated by activated p rsk. thus, p rsk may have anti-apoptotic activity in sars-cov-infected cells. the sars-cov s protein is able to induce phosphorylation of erk / in hek t cells (liu et al. ). the s-induced protein kinase c (pkc)/erk signaling pathway promotes nuclear factor-kappa b (nf-kb) binding to the cyclooxygenase- (cox- ) promoter. similar results have been reported using the n protein of sars-cov (yan et al. ). sars-cov s protein expression induces release of interleukin- (il- ) via erk and p mapk signaling pathways including activator protein (ap- ) in a cells ). on the contrary, phosphorylation of erk / is downregulated in n protein-expressing cos- cells in the absence of serum (surjit et al. ). thus, viral proteins can potentially upor downregulate phosphorylation of erk / . erk / is observed to be phosphorylated in sars-cov-infected vero e cells (mizutani et al. a ). after treatment with mapk/erk kinase and (mek / )-specific inhibitor (pd ), sars-cov-infected vero e cells exhibit no significant changes in activated caspase- or caspase- . thus, activation of erk / is not sufficient to prevent cell death by sars-cov infection. furthermore, activation of erk / is not necessary to establish persistent infection of sars-cov in vero e cells (mizutani et al. ). the sars-cov s protein induces creb binding to cox- promoter mediated via the phosphatidylinositol -kinase (pi k)/pkc/jnk pathway in hek t cells (liu et al. ). expression of the sars-cov n protein induces phosphorylation of jnk in vero e cells (mizutani et al. d ) and in cos- cells in the absence of serum (surjit et al. ). the phosphorylation level of jun, which is dependent upon activation of jnk, also increases in the absence of serum. the sars-cov n protein can activate ap- , which is composed of homodimers and heterodimers of fos, jun, creb, and activating transcription factor (atf) subunits, in vero and huh cells . the viral accessory proteins, a and a, phosphorylate jnk and jnk in hek t cells (kanzawa et al. ) . overall, viral proteins are able to induce phosphorylation of jnk in several cell lines. sars-cov infection induces phosphorylation of jnk in vero e cells after at least h.p.i. (mizutani et al. a ). the vero e cells begin to show rounding at h.p.i and persistently infected cells are observed after h.p.i (mizutani et al. ) . at this time, jnk, akt, and p mapk are phosphorylated in virus-infected cells. treatment with an inhibitor of jnk (sp ), and pi k (ly ), inhibits the establishment of persistence, whereas treatment with an inhibitor of mek / (pd ) and p mapk (sb ) does not inhibit persistence of infection (mizutani et al. ) . thus, two different signaling pathways of jnk and pi k/ akt are important for the establishment of persistently infected vero e cells (mizutani et al. d . akt, which is also known as protein kinase b (pkb), is phosphorylated at both ser- and thr- residues via the pi k signaling pathway upon stimulation by growth factors, insulin, and hormones (toker ; brazil and hemmings ; scheid and woodgett ; welch et al. ) . the main role of akt is inhibition of apoptosis via phosphorylation of the forkhead transcription factor (fkhr) family, glycogen synthase kinase- b (gsk- b), caspase- , and bcl-associated death protein (bad) (cardone et al. ; cross et al. ; datta et al. ) . interestingly, gsk- regulates phosphorylation of n protein (wu et al. ). the m protein of sars-cov induces apoptosis in both hek t cells and transgenic drosophila ). the m protein-induced apoptosis involves mitochondrial release of cytochrome c protein. in sars-cov-infected vero e cells, ser- of akt is phosphorylated at h.p.i. and maximal phosphorylation is observed at h.p.i. (mizutani et al. b) , after which akt is dephosphorylated. thr- phosphorylation has not been detected in vero e cells. the phosphorylation of ser- of akt by viral infection is inhibited by ly , which is an inhibitor of the pi k signaling pathway. an in vitro kinase activity assay of akt in sars-cov-infected cells indicated that akt is highly phosphorylated only at serine residues, but akt activity is low. therefore, weak activation of akt cannot prevent apoptosis induced by sars-cov infection in vero e cells. the phosphorylation of akt in virusinfected cells is necessary to establish persistence, but akt is not phosphorylated after establishing persistent cell lines (mizutani et al. (mizutani et al. , d , suggesting that activation of pi k/akt is essential for the establishment of persistent infection with sars-cov at points in time before cell death. the above characterizations of akt in sars-cov-infected vero e cells are mainly derived from experiments using confluent cells. when subconfluent vero e cells are infected by sars-cov, cell proliferation is inhibited (mizutani et al. c ). sars-cov infection induces dephosphorylation of a serine residue of akt without phosphorylation in subconfluent cultures. thus, downregulation of akt activity in sars-cov-infected cells prevents cell proliferation. the sars-cov n protein is able to activate nf-kb in vero e cells (liao et al. ). as described above, the s-and n-induced pkc/erk signaling pathway promotes nf-kb binding to the cox- promoter (liu et al. ; yan et al. ) . sars-cov s and n proteins may cause inflammation of the lungs by activating cox- gene expression. the a and a viral accessory proteins enhance nf-kb mediated transcription in hek t cells (kanzawa et al. ) . in contrast, the n protein inhibits interferon production in t cells via inhibition of nf-kb (kopecky-bromberg et al. ). the m protein also suppresses nf-kb activity (fang et al. ) . growth arrest and apoptosis via caspase- and caspase- activities are induced in sars-cov c-like protease ( cl pro )-expressing human promonocyte hl-cz cell line (lin et al. ) . the sars-cov cl pro may increase activation of nf-kb and upregulate cytochrome c oxidase and downregulate hsp- , inducing mitochondrial-mediated apoptosis (lai et al. ). viral papain-like protease (plp) regulates antagonism of irf and nf-kb signaling pathways (frieman et al. ). the a protein of sars-cov has the potential to inhibit cell cycle progression at the g phase in hek , cos- , and vero cells (yuan et al. (yuan et al. , . the c-terminal region of the a protein, which includes a potential atpase motif, is essential to inhibit the cell cycle. the a protein expression reduces cyclin d level and inhibits retinoblastoma (rb) phosphorylation. the p phosphorylation is increased by a expression. the a protein expression also blocks cell cycle progression at the g /g phase in hek , cos- , and vero cells by mechanisms similar to those of the a protein (yuan et al. ) . the n protein is a substrate of cyclin-dependent kinase (cdk) as well as gsk, mapk, and casein kinase ii (surjit et al. ) . the n protein directly binds to cyclin d and inhibits activity of the cyclin d-cdk complex. the n protein also inhibits cdk activity by direct binding to the cdk -cyclin complex, resulting in blocking the s phase progression in cos- and huh cells (surjit et al. ) . therefore, proteins of sars-cov may have the ability to inhibit the progression of the host cell cycle, but further detailed analysis is required in sars-cov-infected cells. sars-cov infection induces apoptotic cell death in vero e cells, via dephosphorylation of stat by p mapk activation, and inactivation of akt, as previously described. recent study suggest that sars-cov triggers apoptosis via protein kinase r (pkr) (krähling et al. ). overexpression of sars-cov proteins can induce apoptosis in variable cell lines. induction of apoptosis by various viral proteins may occur at different stages of the infection cycle. sars-cov cl pro expression in hl-cz cells induces apoptosis via caspase- and caspase- (lin et al. ) . furthermore, cl pro expression in hl-cz cells upregulates proteins located in the mitochondria, but downregulates hsp- , which antagonizes apoptosis-inducing factor (lai et al. ). the sars-cov a protein, localized in the mitochondria of infected cells, increases mitochondrial transmembrane potential, reactive oxygen species production, and caspase- activation, resulting in inducing apoptosis in vero, hek , and huh cells ). orf induces apoptosis via caspase- mediated, er stress and jnkdependent pathways (ye et al. ). sars-cov n protein modulates the tgf-b signaling pathway to block apoptosis of sars-cov-infected host cells (zhao et al. ) . in the absence of serum, the sars-cov n protein can induce apoptosis by activating the mitochondrial pathway , and/or by downregulating erk and akt signaling pathways (surjit et al. ) in cos- cells, but not in hep-g and huh- cells ). the sars-cov s protein and its c-terminal domain (s ) induce apoptosis in vero e cells, but the s , e, m, and n proteins are not able to induce apoptosis in vero e cells . in contrast, the sars-cov m and n proteins can induce apoptosis in human pulmonary fibroblast (hpf) cells . the m protein induces apoptosis through modulation of the akt pathway and mitochondrial cytochrome c release in hek t cells and transgenic drosophila [ ]. overexpression of sars-cov a protein in vero e cells induces apoptosis, mediated through a caspase- -dependent pathway or p mapk waye et al. ; padhan et al. ) . the a protein expression in drosophila induces apoptosis, which could be modulated by cellular cytochrome c levels and caspase activity . the sars-cov b protein induces both necrosis and apoptosis in vero e cells (khan et al. ) . the sars-cov a protein interacts with pro-survival proteins, basal cell lymphoma-extra large (bcl-xl), b cell lymphoma (bcl- ), bcl-w, a , and myeloid cell leukaemia sequence (mcl- ), at the endoplasmic reticulum and the mitochondria, resulting in triggering apoptosis in hek t and vero e cells (tan et al. ). interestingly, the a protein does not interact with the pro-apoptotic members, bcl- associated x protein (bax), bcl- homologous killer (bak), bad, and bcl- interacting domain (bid). however, a mutant virus without the a/ b gene is able to induce extensive cpes in the vero cell line (yount et al. ) , suggesting that the a protein is not a dominant contributor to virus-induced cell death in this cell culture system. the sars-cov n protein downregulates the level of bcl- in cos- cells (surjit et al. ). the sars-cov e protein induces apoptosis in jurkat t cells in the absence of growth factors, but apoptosis is inhibited by overexpression of bcl-xl via interaction with the e protein . apoptosis is also inhibited by overexpression of bcl- in sars-covinfected vero cells (bordi et al. ). in the virus-infected vero cells, downregulation of bcl- and upregulation of bax are observed (ren et al. ) . bcl-xl activation plays important roles in establishing persistent infection of sars-cov (mizutani et al. b) . the n protein upregulates the bcl-xl protein level (mizutani et al. d ). these reports indicate that bcl-xl activation is the key to preventing apoptosis due to sars-cov infection. the other viral proteins localized in the mitochondria of infected cells may also interact with bcl-xl and other prosurvival proteins. western blots are used to analyze signaling pathway proteins of cultured cells infected with sars-cov or transfected with plasmids encoding viral proteins. thus, the kinetics of phosphor-proteins regulating signaling pathways is important for understanding which signaling pathways are activated in virus-infected cells. however, in vivo analysis and amounts of mrna from whole blood or tissues of sars patients are primarily analyzed using dna microarrays. unfortunately, when the level of mrna related to a signaling pathway increases in sars patients, as measured by dna microarray analysis, the results do not suggest activation of particular signaling pathways, due to the analysis being performed on a mixed population of cells. the roles of signaling pathways may be different amongst different cell types. analyses of signaling pathways in virus-infected patients are still difficult to perform for these reasons. however, flow cytometric analysis of cell samples from virus-infected patients provides an improved method for the investigation of signaling pathways in vivo. flow cytometric analysis of phospho-p indicated that augmented p mapk phosphorylation of cd monocytes was associated with suppressed p mapk phosphorylation of cd lymphocytes, suggesting that altered leukocyte p activation contributes to abnormal blood cytokine profiles in sars patients ). analysis of cell apoptosis in sars patients is key to understanding the signaling pathways that regulate apoptosis. in sars patients, lymphopenia caused by depletion of t lymphocytes by apoptosis is a common abnormality ). compared to healthy controls, sars patients have significantly lower lymphocyte and platelet counts and have significantly higher vascular cell adhesion molecule- (svcam- ) levels and soluble fas ligand (sfasl) levels, as determined using elisa (enzyme-linked immunosorbent assay). sars patients also have intracellular activated caspase- fragment levels, as measured using flow cytometry (peiris et al. b) . liver impairment commonly occurs amongst patients with sars, indicating that sars-cov may be localized in the liver (chau et al. ). the pathologic features, perhaps due to apoptosis, are the presence of acidophilic bodies, ballooning of hepatocytes, and mild to moderate lobular activities. the thyroid glands of sars-infected patients show extensive injury due to apoptosis of the follicular epithelial cells and the parafollicular cells, as measured using terminal deoxynucleotidyl transferase-mediated dutp nick end-labeling assay . necrosis is also observed in splenic lymphoid tissue and lymph nodes of sars patients (ding et al. ) . myd -mediated innate immune signaling and inflammatory cell recruitment to the lung in balb/c mice may be required for protection from lethal recombinant mouse-adapted sars-cov infection (sheahan et al. ) . further detailed analysis of apoptosis in cells of sars patients is required, but the initial reports indicate the activation of apoptotic signaling pathways in sars patients. both pro-apoptotic and pro-survival signaling pathways are activated in sars-cov-infected cells (fig. . ) . the balance of activities of signaling pathways is important for determination of cell death or cell survival. in sars patients, analysis of signaling pathways is further complicated because many cell types respond to viral infection. for example, immune cells infected by sars-cov produce and release cytokines, and the cytokines activate other cells. thus, in sars patients, many types of cells are infected by sars-cov, compared with one type of cell used for in vitro experiments. in addition, the viral proteins that interact with cellular proteins in signaling pathways must be further clarified to understand the it is particularly important to determine the viral proteins that are necessary and sufficient to fully activate signaling pathways leading to apoptotic cell death. determining the sars-covinduced signaling pathways in sars patients will enable the development of therapeutic reagents that can inhibit the 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chemokine production by severe acute respiratory syndrome coronavirus a/x and a/x proteins through nf-kappab activation overexpression of severe acute respiratory syndrome coronavirus b protein induces both apoptosis and necrosis in vero e cells protein of severe acute respiratory syndrome coronavirus inhibits cellular protein synthesis and activates p mitogen-activated protein kinase severe acute respiratory syndrome coronavirus open reading frame (orf) b, orf , and nucleocapsid proteins function as interferon antagonists severe acute respiratory syndrome coronavirus triggers apoptosis via protein kinase r but is resistant to its antiviral activity a novel coronavirus associated with severe acute respiratory syndrome proteomic analysis of upregulated proteins in human promonocyte cells expressing severe acute respiratory syndrome coronavirus c-like protease the a protein of severe acute respiratory syndrome-associated coronavirus induces apoptosis in vero e cells altered p mitogen-activated protein kinase expression in different leukocytes with increment of immunosuppressive mediators in patients with severe acute respiratory syndrome mitogen-activated protein kinase-dependent hyperinduction of tumor necrosis factor alpha expression in response to avian influenza virus h n microarray and real-time rt-pcr analyses of differential human gene expression patterns induced by severe acute respiratory syndrome (sars) coronavirus infection of vero cells activation of nf-kappab by the full-length nucleocapsid protein of the sars coronavirus severe acute respiratory syndrome coronavirus c-like protease-induced apoptosis spike protein of sars-cov stimulates cyclooxygenase- expression via both calcium-dependent and calcium-independent protein kinase c pathways the genome sequence of the sars-associated coronavirus signal transduction in sars-covinfected cells tyrosine dephosphorylation of stat in sars coronavirus-infected vero e cells importance of akt signaling pathway for apoptosis in sars-cov-infected vero e cells phosphorylation of p mapk and its downstream targets in sars coronavirus-infected cells jnk and pi k/akt signaling pathways are required for establishing persistent sars-cov infection in vero e cells regulation of p rsk phosphorylation by sars-cov infection in vero e cells characterization of persistent sars-cov infection in vero e cells inhibition of cell proliferation by sars-cov infection in vero e cells mechanisms of establishment of persistent sars-cov-infected cells enhancement of cytotoxicity against vero e cells persistently infected with sars-cov by mycoplasma fermentans constitutive activation of stat in human prostate tumors and cell lines: direct inhibition of stat signaling induces apoptosis of prostate cancer cells cytokine regulation in sars coronavirus infection compared to other respiratory virus infections severe acute respiratory syndrome coronavirus a protein activates the mitochondrial death pathway through p map kinase activation coronavirus as a possible cause of severe acute respiratory syndrome the severe acute respiratory syndrome mitogen-activated protein kinase pathway and its role in interferon signaling identification of severe acute respiratory syndrome in canada orf protein of varicella-zoster virus influences jnk/sapk and p /mapk phosphorylation multiple routes to astrocytic differentiation in the cns apoptosis induced by the sars-associated coronavirus in vero cells is replication-dependent and involves caspase ribosomal s kinase (rsk ) activation requires signals dependent on and independent of the map kinase erk characterization of a novel coronavirus associated with severe acute respiratory syndrome unravelling the activation mechanisms of protein kinase b/akt activation of transcription by ifngamma: tyrosine phosphorylation of a -kd dna binding protein interferon-dependent tyrosine phosphorylation of a latent cytoplasmic transcription factor myd is required for protection from lethal infection with a mouse-adapted sars-cov a single phosphotyrosine residue of stat required for gene activation by interferon-gamma interferon activation of the transcription factor stat involves dimerization through sh -phosphotyrosyl peptide interactions protein kinase r (pkr) interacts with and activates mitogen-activated protein kinase kinase (mkk ) in response to double-stranded rna stimulation identification of an extracellular signal-regulated kinase (erk) docking site in ribosomal s kinase, a sequence critical for activation by erk in vivo role of p mapk and rna-dependent protein kinase (pkr) in hepatitis c virus core-dependent nuclear delocalization of cyclin b japanese encephalitis virus infection initiates endoplasmic reticulum stress and an unfolded protein response the sars coronavirus nucleocapsid (n) protein induces actin reorganization and apoptosis in cos- cells the severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by - - -mediated translocation the nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclin-dependent kinase complex and blocks s phase progression in mammalian cells fgf and stress regulate creb and atf- via a pathway involving p map kinase and mapkap kinase- induction of apoptosis by the severe acute respiratory syndrome coronavirus a protein is dependent on its interaction with the bcl-xl protein mechanisms and enzymes involved in sars coronavirus genome expression protein kinases as mediators of phosphoinositide -kinase signaling a cluster of cases of severe acute respiratory syndrome in hong kong etoposide and cisplatin induced apoptosis in activated raw . macrophages is attenuated by camp-induced gene expression signals from the stressed endoplasmic reticulum induce c/ ebp-homologous protein (chop/gadd ) the a protein of sars-coronavirus induces apoptosis in vero e cells pathology of the thyroid in severe acute respiratory syndrome protein kinase b and rac are activated in parallel within a phosphatidylinositide oh-kinase-controlled signaling pathway in vivo functional characterization of the sars-coronavirus a protein in drosophila glycogen synthase kinase- regulates the phosphorylation of severe acute respiratory syndrome coronavirus nucleocapsid protein and viral replication sars coronavirus induces apoptosis in vero e cells nucleocapsid protein of sars-cov activates the expression of cyclooxygenase- by binding directly to regulatory elements for nuclear factor-kappa b and ccaat/enhancer binding protein bcl-xl inhibits t cell apoptosis induced by expression of sars coronavirus e protein in the absence of growth factors a sars-cov protein, orf- , induces caspase- mediated, er stress and jnk-dependent apoptosis severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice g /g arrest and apoptosis induced by sars-cov b protein in transfected cells sars coronavirus a protein blocks cell cycle progression at g /g phase via the cyclin d /prb pathway g phase cell cycle arrest induced by sars-cov a protein via the cyclin d /prb pathway sars-cov nucleocapsid protein induced apoptosis of cos- mediated by the mitochondrial pathway m and n proteins of sars coronavirus induce apoptosis in hpf cells severe acute respiratory syndrome-associated coronavirus nucleocapsid protein interacts with smad and modulates transforming growth factor-beta signaling acknowledgments i am grateful to drs. shuetsu fukushi, masayuki saijo, momoko ogata, kouji sakai, ichiro kurane, and shigeru morikawa (national institute of infectious diseases) for their comments on this manuscript. this work was supported, in part, by a grant-in-aid from the ministry of health, labor, and welfare of japan and japan society for the promotion of science. key: cord- -kcyao i authors: tan, emily l.c.; ooi, eng eong; lin, chin-yo; tan, hwee cheng; ling, ai ee; lim, bing; stanton, lawrence w. title: inhibition of sars coronavirus infection in vitro with clinically approved antiviral drugs date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: kcyao i severe acute respiratory syndrome (sars) is an infectious disease caused by a newly identified human coronavirus (sars-cov). currently, no effective drug exists to treat sars-cov infection. in this study, we investigated whether a panel of commercially available antiviral drugs exhibit in vitro anti–sars-cov activity. a drug-screening assay that scores for virus-induced cytopathic effects on cultured cells was used. tested were clinically approved compounds from several major antiviral pharmacologic classes: nucleoside analogs, interferons, protease inhibitors, reverse transcriptase inhibitors, and neuraminidase inhibitors. complete inhibition of cytopathic effects of sars-cov in culture was observed for interferon subtypes, β- b, α-n , α-n , and human leukocyte interferon α. these findings support clinical testing of approved interferons for the treatment of sars. s evere acute respiratory syndrome (sars) ( , ) is an infectious disease caused by a newly identified human coronavirus (sars-cov) ( , ) . the disease can produce severe pneumonia with a reported fatal outcome of % to %. currently, no effective drug exists to treat sars-cov infection ( ) . the urgency of the outbreak has led to the empiric use of broad-spectrum antibiotics and antiviral agents in affected patients in several countries ( ) ( ) ( ) ( ) ( ) ( ) ( ) . intensive efforts are under way to gain more insight into the mechanisms of viral replication, in order to develop targeted antiviral therapies and vaccines. developing effective and safe vaccines and chemotherapeutic agents against sars cov, however, may take years. the recent epidemic has shown that knowledge is lacking regarding the clinical management and treatment of infected patients. ribavirin ( ) ( ) ( ) ( ) ( ) ( ) ( ) , oseltamivir ( ) ( ) ( ) , foscarnet ( ) , intravenous immunoglobulin ( ) , and other agents have been used to treat patients. preliminary results from in vitro testing indicate that ribavirin concentrations that inhibit other viruses sensitive to ribavirin do not inhibit replication or cell-to-cell spread of the sars-cov ( ) . however, the u.s. centers for disease control and prevention concluded that further in vitro testing of antiviral drugs on other coronavirus isolates and more information on the clinical outcome of patients treated with ribavirin or other antiviral drugs in controlled trials is needed ( ) . the aim of this study was to investigate whether a panel of currently available antiviral agents exhibit in vitro anti-sars-cov activity. three general antiviral strategies are generally found ( ) : ) direct antiviral effects, ) inhibition of viral entry and replication at the cellular level by targeting virus-related processes, and ) enhancement of host immune response. a total of drugs approved for clinical use in the treatment of viral infections were tested in this study. they are representative compounds from major antiviral pharmacologic classes that are currently commercially available: nucleoside analogs, interferons, protease inhibitors, reverse transcriptase inhibitors and neuraminidase inhibitors. a cell-based assay utilizing cytopathic endpoints (cpe) was set up using vero e cells to screen these antiviral compounds. sars-cov has been shown to infect vero e cells, an african green monkey kidney cell line ( ) , and this remains the only in vitro model of sars-cov infection. the initial screen was followed by a plaque reduction assay to determine the % effective concentration (ec ) of compounds showing positive results. these experiments allow rapid screening of commercially available antiviral agents, enabling those with in vitro evidence of activity to move expeditiously into clinical studies, since safety and pharmacokinetic information in humans is already available for other disease indications. here we report that certain interferon subtypes exhibit in vitro inhibitory activity against sars-cov and are candidates for follow-up studies in animal models and patients to determine their efficacy in vivo. to rapidly identify a pharmacologic agent that could be used to treat sars, a collection of antiviral drugs was tested against sars-cov, the etiologic agent of the atypical pneumonia. to investigate a wide spectrum of potential molecular targets, we decided to cover the entire pharmacologic range of commercially available antiviral agents, including agents not expected to be active against coronaviruses. information on antiviral drugs provided here was obtained from prescribing information sheets or from communications with the manufacturer. nucleoside analogues are a diverse class of compounds; in general, they inhibit viral rna or dna polymerases or other enzymes, interfering with nucleic acid synthesis. in this study, the selected compounds that target dna viruses such as herpes simplex virus (hsv) and varicella-zoster viruses (vzv) were acyclovir, ganciclovir, and foscarnet. ribavirin has activity against a range of dna and rna viruses; in different cell lines, ed ranges from to µg/ml. antiretroviral (hiv) drugs include reverse transcriptase (rt) inhibitors and protease inhibitors. selected hiv nucleoside rt inhibitors studied were zidovudine and lamivudine, while hiv protease inhibitors studied were indinavir, nelfinavir, and saquinavir. the third group of antivirals studied were the neuraminidase inhibitors, both commercially available preparations, zanamivir and oseltamivir were used in this study. interferons were the next major class of antivirals studied. various subtypes of interferon α ( a, b, n , and n , human leukocyte) and β ( a and b) were used. amantadine, an old antiviral compound, was also studied. different terms have been used to express antiviral activity, namely, ec , % effective concentration (ec ), and % inhibitory concentration (ic ); table illustrates the range of activity against selected viruses. tenfold dilutions of the drug were tested to cover a broad range of concentrations above and below inhibitory dosages as reported by the manufacturer for other viralhost combinations. compounds already present in aqueous injections were made up to volume by using hank's buffered saline solution. for tablet and capsule formulations with soluble active ingredients, the outer coat was removed wherever applicable, and the preparation was ground in a mortar and pestle. the contents were dissolved in water, vortexed, and centrifuged thereafter at , g. the required volume was pipetted from the supernatant and diluted accordingly. when the active ingredients were insoluble in water (nelfinavir and saquinavir), the contents were dissolved in dimethylsulphoxide (dmso); care was taken to ensure that the final concentration of dmso in the dilutions would not exceed %. for plaque assays, fivefold drug dilutions were prepared by using growth media as specified below. vero e cells (american type culture collection, manassas, va) were propagated in cm cell culture flasks in growth medium consisting of medium (sigma, st louis, mo) supplemented with % fetal calf serum (fcs; biological industries, kibbutz beit haemek, israel). sars-cov va (an isolate from a sars patient in singapore), which has been previously sequenced ( ) , was propagated in vero e cells. briefly, ml of stock virus was added to a confluent monolayer of vero e cells and incubated at °c in % co for h; ml of medium supplemented with % fcs was then added. the cultures were incubated at °c in % co , and the supernatant was harvested after h; in > % of cultures, inhibition of cpe ( +) in each well was observed with an inverted microscope. the supernatant was clarified at , rpm and then divided into aliquots, placed in cryovials, and stored at - °c until use. all virus culture and assays were carried out in the biosafety level- laboratory at the environmental health institute, according to the conditions set out in biosafety in microbiological and biomedical laboratories ( ) . virus titer in the frozen culture supernatant was determined by using a plaque assay. briefly, µl of virus in -fold serial dilution was added, in duplicates, to a monolayer of vero e cells in a -well plate. after h of incubation at °c in % co , the viral inoculum was aspirated, and ml of carboxymethylcellulose overlay with medium , supplemented with % fcs, was added to each well. after days of incubation, the cells were fixed with % formalin and stained with % crystal violet. the plaques were counted visually, and the virus titer in plaque-forming units per ml (pfu/ml) was calculated. the protocol used was adapted from al-jabri et al. ( ) , and all drugs were tested in quadruplicate. briefly, µl of serial -fold dilutions of the drugs were incubated with µl of vero e cells, giving a final cell count of , cells per well in a -well plate. the incubation period was h at °c in % co , except for the interferons, which were incubated overnight with the cells. ten microlites of virus at a concentration of , pfu/well was then added to each of the test wells. the plates were incubated at °c in % co for days and observed daily for cpe. the end point was the drug dilution that inhibited % of the cpe (cia ) in quadruplicate wells. to determine cytotoxicity, µl of serial -fold dilutions of the drugs was incubated with µl of vero e cells, giving a final cell count of , cells per well in a -well plate, without viral challenge. the plates were then incubated at °c in % co for days and examined for toxicity effects by using an inverted microscope. trypsinized vero e cells were resuspended in growth medium and preincubated with interferons (serial fivefold dilution) in quadruplicate wells in -well plates. the next day, the medium was aspirated, and µl of virus was added to each well at a titer of pfu/well. after incubation for h, the virus inoculum was aspirated, and a carboxymethylcellulose overlay containing maintenance medium and the appropriate interferon concentration was added. after days' incubation, the plates were fixed and stained as described previously. the number of plaques was then counted visually, and the concentration of drug that inhibits % of plaques in each well (ic ) was deter-mined. results were plotted in microsoft excel, and a polynomial of order three was used to approximate the data and extrapolate ic and ic values. high titers of infectious sars-cov, originally derived from a respiratory sample of a sars patient, were propagated on vero e cells. the cpe of sars-cov on vero e was evident within hours after infection (figure ). sars-cov-infected cells display a cpe characterized by the appearance of rounded cells and the destruction of the monolayer. a collection of antiviral drugs was tested in the sars-cov cpe inhibition assay ( table ) . the set of drugs tested included seven interferons, five nucleoside analogs, three protease inhibitors, two rt inhibitors, and two neuraminidase inhibitors. complete inhibition of the cpe was observed for four of the seven interferons in the initial screen when very high viral challenge of pfu/well and a high multiplicity of infection (moi = . ) rate were used. complete inhibition, expressed as cia , was observed for interferon β- b (betaferon) at , iu/ml, interferon α-n (alferon) at , iu/ml, interferon α-n (wellferon) at , iu/ml, and human leukocyte interferon α (multiferon) at , iu/ml. ribavirin also completely inhibited the cpe at , µg/ml ( table ) . none of the other drugs showed complete inhibition of cpe, even at the highest concentration of drug tested (table ) . rebif (ifn-β- a) showed slight inhibition of cpe at , iu/ml, but the inhibition was not complete at the screening virus load of , pfu/well. likewise, roferon (ifn-α- a) showed slight, incomplete inhibition at , iu/ml. because the criteria for ascertaining anti-sars-cov activity in this screen were set at % inhibition of cpe, and as high doses of interferons may result in severe clinical side effects, we chose to conduct further evaluations only in the interferons that showed complete inhibition from initial screen, namely, wellferon, multiferon, betaferon, and alferon. based upon results of the primary screen, the four active interferons and ribavirin were retested at two lower viral challenges, and pfu/ well. all four drugs again showed inhibitory effect, although the cia were dependent on viral loads (table ) . at the lowest viral load the cia were iu/ml for both interferon β- b (betaferon) and human leukocyte interferon α (multiferon); and and iu/ml for interferon α-n (alferon) and interferon α-n (wellferon), respectively. no cytotoxicity of the interferons was observed at or near inhibitory concentrations. ribavirin showed inhibitory activity at all three viral loads, but only at high concentrations of the drug, . - mg/ml. at high concentrations of ribavirin ( . - mg/ml) cytotoxic effects were observed on veroe cells, as has been reported for other cell types ( , ) . as such, we consider ribavirin to be inactive against sars-cov. a plaque reduction assay format with pfu of sars-cov (moi = . ) was conducted to determine the ic for betaferon, alferon, and multiferon, the three compounds that showed greatest potency for inhibition of cpe. additional supply was not available for testing interferon α-n (wellferon), as production of this drug has been discontinued. cells were preincubated for h with fivefold dilutions of drug. viral-induced plaques, which developed in days, were counted to determine the inhibitory effect of the drugs at various concentrations. all three interferon preparations displayed a dose-dependent inhibition of sars-cov plaque formation in this assay ( figure ). the ic and ic were determined to be . and iu/ml for betaferon, . and iu/ml for alferon, and and iu/ml for multiferon. betaferon, alferon, multiferon, wellferon, and ribavirin inhibited cpe in sars-cov-infected vero e cells, in decreasing order of potency. ribavirin, a drug widely used in initial efforts to manage sars infections, inhibited cpe completely at - , µg/ml at virus loads of - , pfu per well. the concentration range observed is much higher than concentrations that inhibit other viruses (respiratory syncytial virus, ed - µg/ml, hiv or resistant strains of rhinovirus, - µg/ml), including viruses that were tested on vero cells (west nile virus, new york isolate µg/ml, and uganda isolate µg/ml) ( ) . in addition, the cpe , , , , , , , , , , ( , ) . we observed slight cytotoxicity by microscopic examination of the cells, making it difficult to accurately obtain in vitro efficacy data against sars-cov. it appears that due to the low activity of ribavirin in vitro, inhibitory doses may not be achievable clinically. it is possible that ribavirin would be more effective in combination with interferons. combination therapy with ribavirin and interferon α has now become standard treatment for chronic hepatitis c ( ) ( ) ( ) . additionally, we have tested the effect of ribavirin and betaferon in combination (range of concentration of ribavirin, - µg/ml; range of concentration of betaferon, . - iu/ml). at , pfu, this combination did not demonstrate observable synergistic inhibitory effect against sars-cov. this study describes in vitro activity of four interferon subtypes against the sars-cov. interferons have been used as anticancer and antiviral agents, in particular, for treating hepatitis b and c infections. various groups have reported the clinical benefit of intranasally administered interferon α in human volunteers before and after inoculation with non-sars coronaviruses ( ) ( ) ( ) . the antiviral activity of interferons is mediated by direct effects on infected cells or by modulating an immune response ( ) . interferons interact with specific surface cell receptors, leading to production of interferon-stimulated gene products such as ′ ′-oligoadenylate synthase and protein kinase pkr ( ) . in sars-cov infection, a convenient starting point for the use of interferons against a sars-cov infection would be the usual clinical doses for the treatment of hepatitis b or c. common clinical dosages for interferon α range from to million iu three times a week to million iu daily. for interferon β, data regarding efficacy in the treatment of hepatitis c are conflicting, and interferon β (at doses of to million iu three times weekly) is usually only used in the treatment of infections in patients whose condition no longer responds to other therapies. plasma levels of interferons administered through the subcutaneous route are usually low with correspondingly short half-lives. in view of their mechanism of action, absolute serum levels may not be meaningful as a measure of the biologic activity of interferons, compared to the induction of cellular products such as ′ ′ oligoadenylate synthase. interferon activity varies among different cell types ( , ) , however. specific interferon subtypes which inhibit sars-cov in vero cells may not necessarily have the same effect in other cells; the converse may also be true-that those drugs that are negative in vero cells may be effective in other cell types. we are currently identifying other in vitro models of sars-cov infection that will enable us to address cell-type specific drug effects. also, interferon subtypes exhibited different activity against sars-cov in this study. the mechanism for the difference in activity is unknown. among the products tested, the source of interferon and amount of glycosylation differ. some preparations were derived from human lymphoblastoid or leukocyte cells, while others were recombinantly produced in escherichia coli or mammalian cell culture. we do not know the importance of this observation with respect to possible antiviral mechanisms of the interferons against sars-cov or potential clinical implications of these differences. this study describes rapid screening of commercially available compounds for extension into in vivo research. evidence of activity and data from in vitro studies, however, cannot be easily correlated with clinical performance but rather present promising candidates for follow-up studies. definite recommendations on anti-sars-cov activity of compounds in humans can only be made in the in vivo setting. in conclusion, interferon β- b, α-n , α-n , and human leukocyte interferon α exhibit antiviral activity in an in acute respiratory syndrome in chinaupdate : disease outbreak reported: geneva: the organization a major outbreak of severe acute respiratory syndrome in hong kong a novel coronavirus associated with severe acute 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interferons regulation of interferon alpha responsiveness by the duration of janus kinase activity antiviral activity against transmissible gastroenteritis virus, and cytotoxicity, of natural porcine interferons alpha and beta antiviral activity of interferon against transmissible gastroenteritis virus in cell culture and ligated intestinal segments in neonatal pigs we thank edison liu for his critical review of the manuscript, and the pharmaceutical companies that provided compounds for the assay experiment: roche, novartis, glaxosmithkline, merck sharpe and dohme, serono, schering ag, schering-plough, viragen, hemispheryx, and astrazeneca.ms. tan is a pharmacist with a special interest in pharmacology and clinical research. she was scientific manager at the singapore cancer syndicate, genome institute of singapore, and is now clinical research manger at pfizer ltd. in singapore. key: cord- -kc loqyj authors: osada, naoki; kohara, arihiro; yamaji, toshiyuki; hirayama, noriko; kasai, fumio; sekizuka, tsuyoshi; kuroda, makoto; hanada, kentaro title: the genome landscape of the african green monkey kidney-derived vero cell line date: - - journal: dna res doi: . /dnares/dsu sha: doc_id: cord_uid: kc loqyj continuous cell lines that originate from mammalian tissues serve as not only invaluable tools for life sciences, but also important animal cell substrates for the production of various types of biological pharmaceuticals. vero cells are susceptible to various types of microbes and toxins and have widely contributed to not only microbiology, but also the production of vaccines for human use. we here showed the genome landscape of a vero cell line, in which , putative protein-coding genes were identified in the . -gb genome sequence. a homozygous ∼ -mb deletion on chromosome caused the loss of the type i interferon gene cluster and cyclin-dependent kinase inhibitor genes in vero cells. in addition, an ∼ -mb loss of heterozygosity around this deleted region suggested that the homozygosity of the deletion was established by a large-scale conversion. moreover, a genomic analysis of vero cells revealed a female chlorocebus sabaeus origin and proviral variations of the endogenous simian type d retrovirus. these results revealed the genomic basis for the non-tumourigenic permanent vero cell lineage susceptible to various pathogens and will be useful for generating new sub-lines and developing new tools in the quality control of vero cells. continuous cell lines that originate from mammalian tissues serve as not only invaluable tools for life sciences, but also important animal cell substrates for the production of various types of biological pharmaceuticals. one lineage of the most frequently utilized mammalian cell lines for these purposes is the vero cell lineage, which was established from the kidney tissue of an african green monkey (agm). the primary culture of this tissue was started on march in chiba university in japan, several continuous cell sub-lines were obtained after passages for several months, and a sub-line was then chosen as the standard vero cell line. , vero cells were found to be highly susceptible to various types of viruses including simian polyoma virus sv- , , measles virus, rubella virus, , arboviruses, , and adenoviruses soon after their establishment, and were later found to be also susceptible to bacterial toxins including the diphtheria toxin, heatlabile enterotoxins, and shiga-like toxins (or 'vero' toxins). , after their global distribution, , the application range of vero cells extended from virology in academic laboratories to diagnostic practices in hospitals and bacterial toxin assays. vero cells have pseudo-diploid karyotypes and are non-tumourigenic when a cell passage was not prolonged. , therefore, the vero cell lineage has been successfully utilized as a cell substrate for human vaccines. , vero cells are still the first choice cell model for various types of life-threating emerging pathogens such as h n influenza virus, ebola haemorrhagic fever virus, and middle east respiratory syndrome (mers) coronavirus. animal substrates for vaccine manufacturing are desired to be shifted from animals and eggs to assured continuous cell lines, because animal materials have several concerns related to quality control, stable supply, and animal ethics. - in addition, antigenic drift affecting the vaccination efficacy to humans, which often occurs during the proliferation of influenza viruses in hen eggs, might be improved by shifting to vaccine strainsusceptible human or non-human primate culture cells. therefore, the vero cell lineage should be fully characterized using modern technologies to prepare threats of infectious diseases. the whole-genome sequences of continuous cell lines provide invaluable basic information for various purposes. the genome sequence of a cell line is a comprehensive basis for many genetic characteristics of the cell line, it is closely relevant to other omics approaches such as transcriptomics and proteomics on the cell line, and also facilitates targeted genome editing of the cell line. - however, the whole genome of vero cells has not yet been determined. we here provided a draft sequence of the whole genome of the vero cell lineage after massively parallel sequencing of the genome dna and also karyological and rna-seq analyses. the genome landscape gave a mechanical insight into events that had occurred during the establishment of the permanent cell line susceptible to various types of microbes. in addition, we presented a proof of concept for the genomic-based quality control of cell lines. metaphase chromosomes from vero cells and agm peripheral blood mononuclear cells (pbmc) were analysed by conventional giemsa-banding (g-banding) and multi-colour fluorescence in situ hybridization (m-fish) with differentially labelled human chromosome-specific painting probes ( xcyte kit metasystems, altlussheim, germany). for detailed information, see supplementary data. genome dna was prepared from vero cells (with passage number ) and pbmc using the qiagen blood & cell culture dna kit (qiagen gmbh, hilden, germany). libraries constructed for paired ends and mate pairs were sequenced with hiseq , (illumina inc., san diego, california). after quality filtering, sequences were assembled into scaffolds using sga and sspace software , (see supplementary data for detailed assembly procedure). protein-coding genes were predicted by the augustus program with reference to the human genome as a model and also with rna-seq reads to assist in the predictions. reference genome reads were mapped on the draft genome of the rhesus macaque (macaca mulatta: rhemac ) and agm (chlorocebus sabaeus . : gca_ . ) using the bwa-mem algorithm with default parameter settings. after mapping, potential polymerase chain reaction (pcr) duplicates, which were mapped to the same positions of the reference genome, were removed using picard software (http://picard.sourceforge.net). the average genome coverage of paired-end sequences after removing the pcr duplicates was -fold for the agm reference. single-nucleotide variants (snvs) were called following the best practice pipeline of the genome analysis toolkit (gatk) software package, which includes base quality score recalibration, insertion/deletion (indel) realignment, and discovering and filtering snvs and indels. . . detection of genomic rearrangements in the vero jcrb cell line copy number variants were detected using the control-freec software with a -kb window size and -kb step size. sites with map quality scores , were not used in the analysis. structural variants were identified using the integrated structural variant prediction method delly. junction sequences with ! % identity to the other part of the reference genome and split-read coverage . were also filtered out. to reduce rare and false-positive variant calls, we further applied the following conservative criteria. to detect deletions and inversions, we counted reads spanning non-rearranged sequence regions with at least bp overlapping to each sequence proximal and distal to the boundaries. the number of these canonical reads should be proportional to the number of nonrearranged cells. the number of canonical reads was calculated for each non-rearranged region and divided by , because one rearrangement had two non-rearranged regions. we selected the regions at which rearranged reads (split reads) consisted of at least % of total reads mapped on boundary regions (sum of canonical and split reads). we also filtered out the regions that had , paired-end supports. for additional information, see supplementary data. genome landscape of vero cells [vol. , loss-of-heterozygosity (loh) regions were identified using -mb-size windows with average heterozygosity , . and the ratio of homozygous to heterozygous snvs smaller than . . the cut-off criteria were determined using the distribution of these values in a whole genome ( supplementary fig. s ). the windows were progressively merged into larger regions when average statistics in the region satisfied the criteria. procedures for cell culture, tumourigenicity test, rna-seq, phylogenetic analysis, and genomic pcr are described in supplementary data. all animal experimental procedures were approved by the national institute of biomedical innovation committee on animal resources as the institutional animal care and use committee. the short reads and assembled draft genome sequence have been deposited in the public database (accession number: dra ). the full-length simian endogenous retrovirus sequences obtained in vero jcrb cells have been deposited in ddbj (accession number: ab ). to obtain the reference genome sequence of the cell lineage, cell seeds with the least passage levels were desirable as material. we chose a cryopreserved cell lot registered at the japanese collection of research bioresources cell bank, which, to the best of our knowledge, is the oldest or nearly the oldest lot (with a passage level of from the original primary culture started in march ) among the currently available stocks. lot jcrb (hereafter referred to as vero jcrb ) was expected to be a close relative to widely distributed vero cell seeds such as atcc ccl and who vero - . , , , , - the heteroplantation of vero jcrb cells in immunocompromised nude mice (n ¼ ) did not produce any discernible tumours, while that of human cervical carcinomaderived hela cells produced tumours in nude mice at % efficacy. thus, vero jcrb cells are nontumourigenic, which is consistent with previous findings of vero cells being non-tumourigenic when their passage number was limited. , when choosing the vero jcrb cell seed in this study, we confirmed that there was no microbial contamination in the seed using conventional tests. in addition, dna-seq short reads, which were obtained to resolve the draft sequence of the whole vero cell genome, were employed to comprehensively detect microbe-relevant sequences with a megablast search. no discernible sign of microbe-relevant sequences (except for endogenous retroviral sequences as shown below) was detected in the cell genome sample, which confirmed the absence of microbial contamination in the vero cell seed. the vero jcrb cell line had different karyotypes with chromosomes numbering between and , and the modal chromosome number appeared to be chromosomes in of metaphase cells (fig. a) . the same analysis on pbmc from normal female agm showed chromosomes ( supplementary fig. s a) . because the g-banding karyotypes of vero cells include several abnormal chromosomes (fig. b) , m-fish was applied to identify chromosomal rearrangements. human m-fish probes hybridized efficiently to agm chromosomes and showed syntenic blocks in normal agm karyotypes (fig. c ). this homology to humans was consistent with previous findings, and implied that the human m-fish signal pattern can be used as a normal agm reference (fig. c ). g-banding and m-fish analyses revealed that of vero metaphases represented a main clone with chromosomes. m-fish identified segments in the vero metaphases ( fig. d) , which indicated the occurrence of one fusion and seven translocations. the fusion occurring between chromosomes and led to a reduction in the chromosome number from to . duplications and deletions were also detected in chromosomes (fig. d) , showing rearrangements involved in chromosomes. however, major chromosomal rearrangements were not detected in the other chromosomes by g-banding or m-fish ( fig. b and d) , which suggested that a haploid chromosome set was retained in its original form. in addition to these common features, additional abnormalities were detected in of cells ( supplementary fig. s c and d). although the other eight karyotypes were found to have similar abnormalities, the vero cell line appeared to include several subclones. the main clone accounted for less than half of the population ( of cells) and the vero cell line had a high cytogenetic heterogeneity. two paired-end and three different size mate-pair libraries were constructed from vero cell dna, and the libraries were sequenced by massively parallel sequencing. the insert size distribution and read length of the libraries are summarized in supplementary table s . after quality filtering, we obtained a total of . billion paired-end and million mate-pair reads. the filtered sequences were used for de novo assembly of the vero genome. our final scaffolds consisted of , scaffolds, which were . gb in total, and had the n of kb and n of kb. the augustus program identified , putative proteincoding genes in the genome scaffolds, using rna-seq data as a support. the vero cell lineage was originally reported to be established from the kidney of the agm cercopithecus aethiops. , however, the species classification of agms is a still debated issue and has been revised several times. , in current nomenclature, cercopithecus aethiops is further classified into four different species of chlorocebus. to clarify the species origin of the vero cell lineage, we compared our reads with four previously reported complete mitochondrial sequences of chlorocebus species: chlorocebus aethiops, chlorocebus tantalus, c. sabaeus, and chlorocebus pygerythrus. the mutation rate in the mitochondrial genomes of old world monkeys is known to be markedly higher than that in nuclear genomes ; therefore, the mitochondrial sequences of these four species had sufficiently diverged to cause mapping bias of short reads. we mapped the paired-end reads to all four chlorocebus mitochondrial genomes as a reference and found that the mitochondrial genome of c. sabaeus had the highest coverage and lowest divergence to the mitochondrial genome of vero cells (supplementary table s ). furthermore, a phylogenetic tree using whole mitochondrial genome ( ) could not be distinguished using human m-fish probes, the copy number analysis revealed a gain at chromosome q (fig. b) , which indicated that der( ) had additional material over chromosome . genome landscape of vero cells [vol. , sequences indicated that the vero cell line was closest to c. sabaeus (fig. a) . the gender of the agm individual from which the vero cell lineage was established has not yet been clearly described. a pair of x chromosomes in karyotyping (fig. ) and the almost diploid copy number of x chromosomes in sequence data (fig. b) were observed in vero jcrb cells. collectively, we concluded that the vero cell lineage had been established from a female individual of c. sabaeus. to characterize snvs in the vero cell line nuclear genome, we mapped our paired-end reads to the reference genome of the rhesus macaque (m. mulatta), which has been annotated more than other nonhuman primate genome sequences. a total of % of the unambiguous sites of the rhesus macaque draft genome was covered with high-quality reads, and the genotype was called with high confidence (genotyping quality score ! and coverage ! ), which indicated that the rhesus macaque genome would work as a reasonable reference sequence for analysing the vero cell genome. approximately . million snvs were identified (supplementary table s ). of these, . million and . million snvs were homozygous and heterozygous, respectively. most of the homozygous snvs in the vero cell line were attributed to evolutionary divergence between the genus macaca and chlorocebus, which was - million years ago. , the estimated divergence between the rhesus macaque and vero cell line genomes was . %. the level of heterozygous snvs in the vero cell line was also high and was markedly higher than that in a human individual. this may be partly explained by the higher genetic diversity within agm populations. we also identified . and . million small deletions ( bp) and insertions ( bp), respectively (supplementary table s ). vero cell line by comparing our paired-end reads with the publicly available draft genome of the agm c. sabaeus (c. sabaeus . : gca_ . ), we examined potential . kb-scale deletions, segmental duplications, inversions, and translocations using the information of improper paired-end mapping and split-read mapping. in order to present a landscape of genomic rearrangements in the main population of the vero cell lines, we set stringent criteria for finding genome rearrangements that could identify rearrangements of high frequency in the cell population. a total of deletions, duplications, and inversions of high frequency were detected ( fig. b; supplementary table s ), whereas none of the translocation candidates were detected at a high frequency. agm chromosome harboured a homozygous deletion that spanned across nearly mb (supplementary tables s and s ). the region was syntenic to human chromosome and rhesus macaque chromosome (from mllt to lingo ) and contained genes including the type i interferon gene cluster and cdkn genes ( table s ). this homozygous large deletion was validated as given below. we identified copy number variations in different genomic regions using the mapping coverage of paired-end reads. nine regions in chromosomes q, q, q, q, , q, , and xq had three copies, while eight regions at p, p, p, q, q, q, and xq were identified as a single copy (fig. b) . agm chromosome p showed an intermediate coverage between two and three copies, in which the duplication and translocation of agm chromosome p varied among the clones. these large-scale copy number changes agreed well with the karyotypes examined using m-fish analysis (fig. d) . loh regions were identified using the density of snvs and its ratio to the density of homozygous snvs. in addition to the identified haploid regions, we identified large loh blocks in chromosomes q, q, and . the loh on chromosome harboured a large homozygous deletion ( fig. c; supplementary table s ; see also below). the large deletions predicted by the massively parallel sequencing system were validated by genomic pcr. regarding the . -mb deletion of chromosome , a set of pcr primers striding across the deletion junction produced a -bp amplicon from vero cells, but not from normal agm cells, while a pcr primer set designated for the agm genome produced a predicted amplicon from normal agm cells, but not from vero cells (fig. a) . we tested two vero cell lines, jcrb and atcc ccl , and obtained identical results (fig. a) . in similar validation tests for another four large (. kb) predicted deletions (supplementary table s ), dna fragments with breakpoint junctions were amplified from the vero cell lines, but not from agm pbmc for all these deletions, which confirmed the existence of these deletions in vero cells ( fig. a; supplementary fig. s ). four small ( - kb) predicted deletions were also confirmed to exist ( fig. b; supplementary fig. s ). analysis of collected srv-related short reads from all paired-end short reads of the vero jcrb cell line, followed by analyses of gene assignment and long terminal repeat (ltr) finding, identified the , bp complete srv genome sequence. the medians of variant frequencies were . and . % in the highly variable and env-deleted region (nucleotide position - ) of srv, respectively (fig. ) . the copy number of srv with . -kb full length was estimated to be %, while that of srv with the deletion of the - nucleotide (nt) region encompassing the c-terminal part in env and a portion of ltr was %. the srv-vero of jcrb had % of the same nucleotides as those of atcc ccl (genbank_id: jn ). the number of minor alleles was for snvs and for the insertion among the whole complete consensus srv sequence in the vero jcrb genome. four minor mutation sites caused three nonsense mutations and one frameshift on the pol or env region (supplementary table s ). previous studies suggested a frameshift mutation in pol (position ) or frameshift mutation in prt (its position was not reported) on the srv proviral sequences of vero e or atcc ccl cells. , however, our study did not detect equivalent mutations on the srv proviral sequences of vero jcrb cells; however, other notable mutations were instead detected (supplementary table s ). by comparing with the agm reference genome, the whole genome structure of vero cells provided various important insights into the molecular characterization of this cell line. vero cells are incapable of producing type i interferon in response to viral infections, which may be the main cause for the high susceptibility of these cells to various types of microbes. the homozygous deletion of aand b -interferon genes in vero cells was previously reported using classical dna hybridization analysis. the present study determined an -mb deleted region in chromosome at the nucleotide level and further revealed an -mb loh around the deleted region ( fig. b and c) , which suggested that an -mb deletion first occurred in one of two homologous chromosome during the establishment of cells, followed by a large-scale conversion that fixed the homozygous deletion of the region in the vero cell lineage. the deleted region in chromosome of vero cells is syntenic to human chromosome p -p , which contains many genes of type i interferons (a , a , a / , a , a , a , a , a , v , and b ) (supplementary table s ). the human syntenic region corresponding to the deleted region in agm chromosome also contained cdkn a and cdkn b: cdkn a encodes cyclin-dependent kinase (cdk) inhibitor a/p ink a (which inhibits cdk , a negative regulator of the retinoblastoma protein prb) and p arf (which inhibits the p -negative regulator mdm ) in an alternate reading frame to the former, while cdkn b encodes cdk inhibitor b/p ink b (which inhibits another prb-negative regulator cdk ) (supplementary table s ). , the cdk inhibitors and mdm inhibitor act as key regulators of the cell cycle, and mutations in cdkn a-cdkn b often occur in various types of human cancer; however, these mutations by themselves are not enough to transform genome landscape of vero cells [vol. , cells into tumourigenic cells. - the loss of both cdkn a and cdkn b may play a crucial role in the acquirement of immortality in the vero cell lineage. originally non-tumourigenic vero cells may then acquire tumourigenicity when additional unknown mutations accumulate during prolonged passages. although the karyological analysis demonstrated that vero cells had various chromosomal rearrangements (fig. c) , no translocation was identified in the wholegenome sequence (fig. b) . this discrepancy may have been due to technical limitations. the karyotyping results obtained showed that most of the translocation events occurred between the telomeric regions of chromosomes, which could not be identified by sequencing if chromosomes fused via repeat sequences. in addition, in order to filter out rare chromosomal rearrangements in a cell population, events that occurred in only one of the homologous chromosomes may not be identified in our filtering criteria. therefore, the absence of translocation rearrangements by sequencing does not contradict the results of the karyological analysis, which showed that all or most chromosomal translocations were observed in one of the two homologous chromosomes (fig. c and d; supplementary fig. s c and d) . haplotype sequencing may be necessary to determine such heterozygous events. many srv sequence variations existed in vero jcrb cells (fig. ) . as for srv associated with the vaccine-producing vero e cell line (the parental cell line of which is atcc ccl ), a frame-shifting single-nucleotide insertion in the polymerase gene was identified. this frameshifting mutation was not detected in srv associated with the vero atcc ccl cell line or vero jcrb cell line (this study). srv variant sequences lacking the u and r regions of ltr were instead detected in vero atcc ccl -associated srv, while the results of this study suggested that some srv copies in the vero jcrb cell genome were defective in the env- ltr region (fig. ) . thus, a large amount of diversity may occur in proviral srv sequences during the passage of vero cells. various quality tests must be conducted in order to fully characterize cell banks for pharmaceutical use (the who guidelines on animal cell substrates are available at http://www.who.int/biologicals/vaccines/ trs_ _annex_ .pdf.) many of these tests rely on conventional methodology, and some tests still use many experimental animals. the whole-genome sequence should be invaluable reference information to develop more rational and effective methods for identification, genetic stability, and microbial agents in pharmaceutical cell banks. for example, the currently authorized tests for cell identity consist of classical methods (e.g. isoenzyme analysis and g-band analysis) and more modern dna profiling methods (e.g. restriction fragment length polymorphism and variable number of tandem repeats analysis). these tests, even if not all, require a considerable amount of time and money as well as well-trained technical skills, and some are not accurate enough to discriminate different cell lines established from the same biological species. this study presented a proof of the concept that pcr analysis will open a rapid and accurate alternative method for the cell identity test to detect unique chromosomal deletions (fig. ) . metagenome analysis is a powerful approach that can be used to survey microbial contamination in biological pharmaceuticals. , , this study also employed dna-seq short reads of vero cell genome dna to comprehensively survey microberelated sequences in the cell sample, and detected no discernible sign of microbe-relevant sequences (except for endogenous srv) in vero jcrb cells. in this direction, it is crucial to distinguish between endogenous and exogenous viral-like sequences, because the appearance of the former is inevitable and can serve as an internal positive control in metagenomic analysis for microbial agents. in addition, the heterogeneity in srv sequences as discussed above may also be a good genomic signature for identifying a specific cell seed among various vero cell sub-lines. the whole-genome genome landscape of vero cells [vol. , sequence of vero cells will also be an invaluable resource for engineering the specific genes of cells by recently advanced genome-editing technologies. in conclusion, this study showed the genomic characteristics of vero cells, which have been a good cell model for microbial infection for a long time. in addition, the genome landscape will be a crucial resource not only for the quality control of vero cell lines, but also for the development of novel sub-lines in the future. studies on sv in tissue culture: preliminary step for cancer reserach in vitro studies on sv in tissue culture: preliminary step for cancer research in vitro studies on measles virus. ii. propagation in two established simian renal cell lines and development of a plaque assay cytopathic and plaque assay of rubella virus in a line of african green monkey kidney cells (vero) replication of rubella virus in a continuous line of african green monkey kidney cells (vero) characterization of the tacaribe group of arboviruses. i. propagation and plaque assay of tacaribe virus in a line of african green monkey kidney cells (vero) biological characteristics and viral susceptibility of an african green monkey kidney cell line (vero) micro cell culture method for determination of diphtheria toxin and antitoxin titres using vero cells. i. studies on factors affecting the toxin and antitoxin titration assay of escherichia coli heat-labile enterotoxin with vero cells vero response to a cytotoxin of escherichia coli sporadic cases of hemorrhagic colitis associated with escherichia coli o :h cell line vero deposited to japanese cancer research resources bank the lineage of the vero, vero and its clone c in the united states cytogenetic examination of vero cells derived from the present stock tumorigenicity of vero cells qualification of working cell banks for the vero cell line to produce licensed human vaccines vero cell platform in vaccine production: moving towards cell culture-based viral vaccines cell culture-derived influenza vaccines from vero cells: a new horizon for vaccine production strategies for developing vaccines against h n influenza a viruses ebola and marburg viruses: ii. their development within vero cells and the extra-cellular formation of branched and torus forms isolation of a novel coronavirus from a man with pneumonia in saudi arabia low - influenza vaccine effectiveness associated with mutation in the egg-adapted h n vaccine strain not antigenic drift in circulating viruses talens: a widely applicable technology for targeted genome editing rna-guided human genome engineering via cas multiplex genome engineering using crispr/cas systems improvement of trypsin method for banding chromosomes scaffolding pre-assembled contigs using sspace efficient de novo assembly of large genomes using compressed data structures augustus: a web server for gene finding in eukaryotes fast and accurate short read alignment with burrows-wheeler transform the genome analysis toolkit: a mapreduce framework for analyzing next-generation dna sequencing data control-freec: a tool for assessing copy number and allelic content using next-generation sequencing data history of vero cells in japan regulation of the interferon system: evidence that vero cells have a genetic defect in interferon production absence of viral sequences in the who-vero cell bank. a collaborative study evolutionary conservatism in arrangement of genetic material. a comparative analysis of chromosome banding between the rhesus macaque ( n equals , arms) and the african green monkey reciprocal chromosome painting shows that the great difference in diploid number between human and african green monkey is mostly due to non-robertsonian fissions assessment of the diversity of african primates mitochondrial diversity and distribution of african green monkeys (chlorocebus gray, ) a challenge to the ancient origin of sivagm based on african green monkey mitochondrial genomes mitochondrial-nuclear interactions and accelerated compensatory evolution: evidence from the primate cytochrome c oxidase complex catarrhine primate divergence dates estimated from complete mitochondrial genomes: concordance with fossil and nuclear dna evidence a molecular phylogeny of living primates a map of human genome variation from population-scale sequencing delly: structural variant discovery by integrated paired-end and split-read analysis viral nucleic acids in live-attenuated vaccines: detection of minority variants and an adventitious virus ensuring the safety of vaccine cell substrates by massively parallel sequencing of the transcriptome defectiveness of interferon production and of rubella virus interference in a line of african green monkey kidney cells (vero) homozygous deletion of the a-and b -interferon genes in human leukemia and derived cell lines the regulation of ink /arf in cancer and aging epigenetic regulation of the ink b-arf-ink a locus: in sickness and in health diverse mechanisms of somatic structural variations in human cancer genomes evaluation of cell substrates for the production of biologicals: revision of who recommendations chemical induction of endogenous retrovirus particles from the vero cell line of african green monkeys key: cord- -i cfmw x authors: peng, ju-yi; horng, yi-bing; wu, ching-ho; chang, chia-yu; chang, yen-chen; tsai, pei-shiue; jeng, chian-ren; cheng, yeong-hsiang; chang, hui-wen title: evaluation of antiviral activity of bacillus licheniformis-fermented products against porcine epidemic diarrhea virus date: - - journal: amb express doi: . /s - - - sha: doc_id: cord_uid: i cfmw x bacillus licheniformis (b. licheniformis) is commonly used as probiotic and its secondary metabolites are attractive anti-microbial candidate. in the present study, we aimed to evaluate the antiviral activity of crude extracts from b. licheniformis against porcine epidemic diarrhea virus (pedv), a highly contagious enveloped porcine virus that has caused great economic loss in pigs. in vivo, pedv-infected piglets supplemented with air-dried solid state fermentative cultivate containing b. licheniformis-fermented products (blfp) showed milder clinical symptoms and decreased viral shedding. importantly, no significant systemic pathological lesions and no reduction in average daily gain were noted in pigs supplemented with the blfp, which suggests that it is safe for use in pigs. in vitro experiments revealed that while b. licheniformis crude extracts exhibited no toxicity in vero cells, co-cultivation of b. licheniformis crude extracts with pedv significantly reduced viral infection and replication. summarized current results suggest that the b. licheniformis-fermented products could be a novel candidate food additive for reducing the impact of ped on the swine industry. beginning in , outbreaks of new variants of porcine epidemic diarrhea virus (pedv) have caused high mortality and morbidity in piglets, leading to great economic losses (lee ) . the virus causes porcine epidemic diarrhea (ped), which is characterized by watery diarrhea, vomiting, and severe dehydration in pigs of all ages, especially suckling piglets (lee ; song and park ) . this highly contagious disease has spread quickly in porcine industries in several countries (jung and saif ; lee ) . furthermore, pedv infection causes severe perturbations of gut microbiota, reducing probiotic bacterial abundance, enriching pathogenic bacteria, and even impairing the growth performance of pedv-surviving pigs (alvarez et al. ; deping song et al. ) . the development of effective protective agents against pedv infection is urgently needed. probiotics is one of the choices to be an alternative to antibiotic growth promoter, agp (abudabos et al. ) . our recent studies reported that lactobacillus species and clostridium butyricum-fermented probiotics product alleviate diarrhea incidence and reduce the gut pathogens in weaning piglets ). on the other hand, bacillus subtilis (b. subtilis) and b. licheniformisfermented products could increase growth performance and mitigate clostridium perfringens-induced necrotic enteritis in broilers (cheng et al. ; lin et al. ) . enhancement of nutrient digestibility and lactobacillus counts of feces by feeding b. subtilis and b. licheniformis has also been reported in pigs (lan and kim ) . it has been demonstrated that b. licheniformis exhibiting antimicrobial activity against pathogens might be due to the production of antibacterial biosurfactants (lin et al. ) . furthermore, our experiment results also demonstrated that b. licheniformis-fermented products exhibit antibacterial activities against clostridium perfringens and staphylococcus aureus in vitro (lin et al. ). in the present study, the antiviral effect of the blfp crude extract against pedv were evaluated in pigs. the surfactin-like peptide in the blfp crude extract was identified from the secondary metabolite of b. licheniformis fermentative cultivates. the in vitro toxicity and antiviral ability of the surfactin-like peptide in the blfp crude extract against pedv were evaluated using the vero cells. vero c cells (american type culture collection (atcc) no. crl- ) were maintained in dmem (gibco, ny, usa) supplemented with % fetal bovine serum (hyclone, utah, usa) and % penicillin-streptomycin-amphotericin b (gibco, ny, usa). the passage pedv-pintung strain (pedvpt-p ) viral stock was used at a titer of × tcid /ml. air-dried solid-state fermentative cultivates of b. licheniformis (weigmann) chester (atcc ® ™ ) were suspended in distilled water and heated at °c for min. supernatants were harvested after centrifugation at , rpm for min. after adjusting the ph value to . with n hcl for protein precipitation, the precipitates were dried in a freeze dryer after washing twice with distilled water. this b. licheniformis-fermented products (blfp) was used in animal study. to characterized the blfp, the surfactin derived from bacillus subtilis (sigma-aldrich, st louis, usa) was used as standard substance. the content of blfp in the fermentative product was determined by high-performance liquid chromatography (hplc) as previously described (schneider and marahiel ) . briefly, after filtration, samples were subjected to analysis via spd- a hplc (shimadzu, japan) with a pre-packed lichrospher rp- column (merck, darmstadt, germany). the mobile phase was a mixture of . mm trifluoroacetic acid and ml ddw with ml % methanol. elution was performed at a flow rate of ml/min and determined with a uv detector ( a vp, shimadzu, tokyo, japan) at nm. the gradient strategy was as follows: - . min, % a to % a; . - min, keeping % a and % b (a, acetonitrile; b, ultrapure water); min pressure bar, max pressure bar, pressure stability bar; injection volume μl; syringe speed μl/s; flush volume μl. liquid chromatography-mass spectrometry (lc-ms) full scan positive mode was performed with m/z ranging from to . fifteen -week-old, pedv-fecal rna and pedv seronegative, large white × duroc crossbred pigs were acquired from a conventional pig farm with no known history of ped. treatments were: ( ) control (n = ); ( ) pedv (n = ); and ( ) pedv + blfp (n = ). these pigs were fed a commercial diet mixed with or without kg/l blfp as feed additives for days prior to the viral challenge, and were challenged with or without × tcid of the virulent pedvpt-p (p ) at weeks of age (table ) . each group was housed in a separate fenced area. clinical symptoms, fecal consistency scoring, and fecal viral shedding were recorded and tested daily. the clinical scores were recorded using a -scale: , normal feces; , pasty; , semi-fluid; , fluid. all piglets were weighed weekly and sacrificed days post-infection (dpi) for safety assessment by pathological examination. the animal experiment was approved by the institutional animal care and use committee of national taiwan university (taipei, taiwan, ntu -el- ). real-time quantitative rt-pcr (qpcr) was performed based on a previously described method with modification (chang et al. ) . the viral rna was extracted from μl of culture supernatants using cador ® pathogen qiacube ® (qiagen, chatsworth, ca, usa) according to the manufacturer's instructions. reverse transcription was performed using the quantinova reverse transcription kit (qiagen) according to the manufacturer's protocol. for qpcr assay, the quantinova probe master mix of fecal swabs were used to determine the genomic equivalents (ge). the detection limit of the real-time rt-pcr was ge of dna using the plasmid encoding the pedv n gene as a standard (data not shown). all pigs were euthanized and necropsy was performed at dpi to evaluate the safety of blfp in pigs. representative tissue samples were collected, fixed in % neutral-buffered formalin, processed routinely, sliced into -μm-thick sections, and stained with hematoxylin and eosin. the histopathological observations were recorded and assessed blindly by one veterinary pathologist. to evaluate the cytotoxicity of blfp crude extract in vitro, vero cells were first grown in a -well microplate (corning life sciences, corning, ny, usa) at a density of , cells per well day prior to the experiment. after removing the culture supernatant, ten-fold serially diluted blfp crude extract in pi medium (dmem supplemented with . % tryptose phosphate broth (sigma-aldrich, mo, usa), . % yeast extract (acumedia, michigan, usa), and μg/ml of trypsin (sigma-aldrich, mo, usa) was added to the cell monolayer. after h, the culture supernatant was removed and μl alamarblue ™ was added (g-biosciences, st. louis, usa; % in pbs). after h of incubation at °c, the plate was read with the excitation wavelength at nm and with the emission wavelength at nm. the od value of each well was calculated to obtain the percentage reduction of alamarblue according to the manufacturer's instructions. additionally, an untreated group g , which consisted of vero cells without any treatment, was included to represent % reduction of alamarblue. all data were further calculated as a percentage of the untreated group g . normalized data were plotted against concentrations of blfp crude extract and fitted to a non-linear regression curve using graph-pad prism (graphpad software, san diego, ca). the % cytotoxicity concentration (cc , the concentration of blfpat which cellular viability was reduced to %) was calculated accordingly. to study the antiviral activity of blfp crude extract against pedv, the biosurfactants were added at different time points during the viral infection. vero cells were seeded in a -well microplate ( , cells/well) day before the experiment. blfp crude extract at ppmin pi medium were added to each well in different orders to treat tcid /ml pedv infections in cells. the experiment included five groups (as illustrated in fig. a to further investigate the antiviral mechanism in the post-drug group (g ), the replication kinetics of pedv in vero cells with or without crude extract of blfp were compared. vero cells were seeded in a -well microplate ( , cells/well) day before the experiment. the crude extract of blfp at ppm in pi medium was added to tcid /ml pedv-infected cells. culture supernatants and cells were collected separately at the , , , , and h post-infection. viral titers in culture supernatants were determined using the reed-müench method (reed ) and are expressed as the % tcid /ml. virus-specific rna in culture supernatants and in cells was quantified by real-time reverse transcription-pcr. virus titration was performed in the culture supernatants. briefly, the first -fold dilution and the subsequent -fold serial dilution of the supernatants were added to , cells/well vero cells on -well microplates. after h of incubation, the inoculants were aspirated, and the cells were washed with pi medium two times and then cultured in pi medium. after h, the plate was subjected to cytopathic effect (cpe) observation and the titer of pedv was determined using the reed-müench method (reed ) and is expressed as the % tcid /ml. to determine the ic , vero cells were seeded in -well microplates ( , cells/well) one day before the experiment. ten-fold serially diluted crude extract of blfp in pi medium was added to vero cells h after % tissue culture infectious dose (tcid ) pedv infection. after h, the percent reduction of alamarbluestaining was examined by the alamarblue ™ assay as described above. all normalized data were plotted and fitted to a nonlinear regression curve in graphpad prism (graphpad software) to generate the ic . to elucidate the direct virucidal activity of crude extract of blfp against pedv, the pedvpt-p viral stock at a titer of × tcid /ml was mixed with or without ppm blfp for h at °c. the mixtures were serially diluted -fold in pi medium and added to vero cells. after h, the viral titers were determined as described above. all data were analyzed and plotted using graphpad prism (graphpad software). all error bars represent standard deviation (sd). the significance of the differences among groups in the cell-based study was determined using student's t test or one-way anova with tukey's multiple comparison. a p-value of < . was interpreted as statistically significant. the results of the average daily gain (adg) and fecal rna shedding in the animal experiments were analyzed and the variables among groups were compared using a non-parametrical kruskal-wallis test, with p < . considered significant. all data were analyzed using graphpad prism software (graphpad prism inc.) as shown in fig. , hplc analysis of the b. licheniformis extract showed a single peak (fig. b) at the retention time of - min, which is identical to the standard substance surfactin derived from bacillus subtilis (fig. a) . (fig. ) . to assess the efficacy of the antiviral ability of blfp against pedv, clinical symptoms after viral challenge were recorded daily (fig. ) . the fecal conditions of pigs in all groups were scored as normal (score = ) before pedv inoculation. in general, pigs supplemented with blfp exhibited milder symptom compared to piglets supplemented with controls. in the control group, all five pigs ( / ) exhibited normal clinical signs (score = ) during the study. in the pedv group in which pigs were supplemented with control food, different severities of pedv-associated clinical signs were first detected days post infection (dpi). two of these five pigs ( / ) exhibited semi-fluid feces (score = ) at dpi, while / pigs showed typical semi-fluid (score = ) to watery diarrhea (score = ) at to dpi, and eventually recovered after dpi. in the pedv + blfp group, most of the pigs exhibited pasty feces (score = ) at - dpi. only one pig exhibited transient typical watery diarrhea (score = ) at dpi. compared to the pedv-infected group with control food, the severity and duration of typical diarrhea (scores - ) were reduced in the pedv + blfp group. to quantify pedv-associated fecal viral shedding to evaluate the antiviral efficacy of blfp against pedvpt-p , rt-qpcr was used to detect fecal viral shedding. in the pedv group (the grey line in fig. ) , fecal viral shedding was first detected ( . ± . log ge) at dpi, found to gradually increase to peak viral load ( . ± . log ge) at dpi, and was continuously detected until dpi. in the pedv + blfp group (the black line in fig. ) , the pattern of viral shedding was similar to but lower than that inthe pedv group during the study, although the difference was not statistically significant. all pigs were weighed weekly in order to evaluate their growth performance with and without biosurfactants as a feed additive. no significant difference in the average daily gain (adg) of pigs was noted among all groups in each week (fig. ) . a b fig. clinical scoring of fecal consistency. compared to pedv-infected groups, the appearance and duration of typical diarrhea (score - ) were reduced in the pedv + blfp group to evaluate the safety of blfp as a feed additive for pigs, necropsy was performed in all piglets weeks postinfection for gross and histopathological examination. for the gross and histopathological examinations, no macroscopic or microscopic lesions were noted in any of the groups. these findings suggest that blfp as a feed additives at kg/ton are safe in conventional pigs up to days of feeding. to determine the median cytotoxic concentration (cc ) of blfp crude extract in vero cells, the blfp crude extract was serially diluted -fold from to . ppm and added to cells. h post-incubation (hpi), no cytotoxicity due to blfp crude extract was observed in vero cells (fig. ) . to determine the maximal inhibitory concentration, the post-drug group g was used to evaluate the maximal inhibitory effect of blfp crude extract by cocultivating fig. fecal shedding of pedvpt-p detected in piglets fed < kg/ton blfp in each group (n = ). the pattern of viral shedding in the pedv + blfp group was similar to but lower than that of the pedv group during the study, with no significant difference detected. changes in the mean values of genomic equivalents (ge)/ml are presented as log values ± sd. the viral rna loads of inoculated groups and the control group were compared using a non-parametrical kruskal-wallis test fig. average daily gain of pigs in each group. all pigs were weighed weekly in order to evaluate their growth performance with or without blfp as feed additives. no statically significant difference in the average daily gain was noted among all groups each week blfp crude extract with pedv-infected cells during the whole study. the results indicated that the antiviral activity of blfp crude extract is dose-dependent, and the inhibition of pedv-induced cpe was calculated with an ic value of . ± . ppm (fig. ) . to elucidate the antiviral activity of blfp crude extract against pedv, ppm of blfp crude extract was added to the culture at different time points during the viral infection. as shown in fig. a , typical pedv-induced cpe was characterized by formation of syncytial cells, cell death, and detachment from the culture plate in pedv-infected cells. the percent reductions of alamar-blue staining in g -g -cells treated with blfp crude extract h before infection, at the same time as infection, and h after viral infection, then replaced with fresh pi medium-were calculated as . ± . %, . ± . %, and . ± . %, respectively, and showed no significant dose-dependent effect of blfp crude extract against pedv. the half-maximal inhibitory concentration (ic ) of blfp crude extract against pedv in vero cells is . ± . ppm. data are presented as the mean ± sd out of three test replicates differences from those in the pedv-infected group g (fig. b) . for the post-drug group g , significant inhibition of pedv-induced cpe, with an . ± . % reduction of alamarblue staining, was observed compared to that in g and other groups (g -g ). similarly, the amount of viral rna in supernatants from g , ranging from . to . log ge, was significantly lower than that of g -g , ranging from . to . log ge (fig. c) . additionally, the viral titer of supernatants in the g group, which was less than tcid /ml, was lower than that of g -g , ranging from × to × tcid /ml. moreover, when evaluating the direct virucidal activity of blfp crude extract against pedv, a reduction of the viral titer of pedv from to tcid /ml was observed. to further investigate the antiviral mechanism in the post-drug group (g ), the replication kinetics of pedv in vero cells in the presence or absence of blfp crude extract were determined. compare to pedv-infected vero cells cultured without blfp crude extract, which showed viral titers from . × at hpi to . × tcid /ml at hpi (fig. a) , cells treated with blfp crude extract had an undetectable extracellular viral titer, which was significantly lower than that of cells without blfp crude extract. similarly, extracellular viral rna levels in pedv-infected cells cultured with biosurfactants were significantly lower than those without blfp crude extract and hpi (fig. b) . however, no significant differences in intracellular viral titers (fig. c ) or intracellular viral rna (fig. d) were noted in pedv-infected cells treated with or without blfp crude extract. since late , new variants of pedv have been identified in several counties and have caused a devastating disease and economic loss. this viral threat has prompted a global effort to develop antivirals against new variant pedvs in vitro (song and choi ; kwon et al. ) and in vivo (cho et al. ; kim et al. ) . herein, we demonstrated that: ( ) blfp crude extract presents no cell toxicity to vero cells, and exhibits significant antiviral ability against pedvpt-p in vero cells when biosurfactants are co-cultivated with pedv; ( ) piglets supplemented with blfp at concentrations of kg/ton or less exhibited milder clinical symptoms and lower viral shedding compared to piglets supplemented with control food, and blfp caused no significant toxicity. therefore, blfp is suggested to be a promising novel feed additive that can act as an additive against pedv in the field. the main antiviral mechanism of blfp crude extract is thought to be related to its unique biochemical structure that increases membrane permeability to disrupt and lyse microbial membranes (huang et al. performing a direct-virucidal study of blfp crude extract against pedv, a ten-fold reduction of the viral titer was able to confirm the directed-antiviral ability of blfp crude extract. according to our hplc analysis, a peak of surfactin-like peptide was identified in the blfp crude extract. several different potential antiviral mechanisms of biosurfactants have been previously reported (vollenbroich et al. ; wang et al. ) . lipopeptide biosurfactants have been previously suggested to possess probiotic attributes for animal and human use (schneider ; sen ) . a previous study demonstrated that the antiviral ability of surfactin from b. subtilis was achieved through competition with the tgev entry receptor . the inhibition of viral enzymes such as proton-atpase that are required for the entry of some viruses into cells by surfactin from b. subtilis (vollenbroich et al. ) has also been reported. it is worth noting that not only did co-cultivation of the surfactin-like peptide in the blfp crude extract with pedv-infected vero cells significantly reduce viral infection and replication in the study, but blfp crude extract-treated pedvinfected cells also exhibited an undetectable extracellular viral titer, suggesting that blfp crude extract may play an important role in reducing the release of virions. these results suggest additional mechanisms of blfp crude extract against pedv in vero cells. although further study of the antiviral mechanisms of blfp crude extract is needed in the future, observation of the diverse antiviral activities of blfp crude extract will lead to further development of this new therapeutic candidate. previous studies demonstrated that lipopeptides produced by various bacillus spp. were safe in mice (youn-hwan hwang et al. ) and weaned piglets (torres et al. ) when administered under a certain concentration. in order to use blfp as a feed additive for its antiviral properties, assessment of its antiviral and safety profile in pigs is essential. our results demonstrated that piglets supplemented with blfp show milder symptoms fig. replication kinetics of pedv in vero cells treated with or without blfp crude extract. a extracellular viral titers in the supernatants of pedv-infected vero cells treated with and without blfp crude extract were determined by viral titration in vero cells using the reed-müench method and expressed as the % tcid /ml. b extracellular viral rna levels in the supernatants of pedv-infected vero cells treated with or without blfp crude extract were determined by real-time reverse transcription (rt)-pcr. c intracellular viral titers in pedv-infected vero cells treated with or without blfp crude extract were determined by viral titration in vero cells. d intracellular viral rna levels in the supernatants of pedv-infected vero cells treated with or without blfp crude extract were determined by real-time rt-pcr. statistical analysis was performed using student's t-test, and statistically significant differences were labeled with *(p < . ) and have reduced viral shedding. importantly, no significant systemic pathological effects and no changes in average daily gain were noted. these results suggest that the biosurfactants at concentrations of kg/ton or less will be very safe in pigs in future applications. in a previous study, it was found that administration of surfactin from b. subtilis at over kg/ton leads to necrosis of hepatocytes in rats (hwang et al. ). in the future, animal experiments using higher dosages of biosurfactants would be necessary to validate its maximal safety dosage. in the present study, blfp was investigated as an antiviral candidate against pedv. in vitro, blfp crude extract exhibited antiviral activities against pedv when incubated long-term with pedv-infected cells. in vivo, we first validated the safety and antiviral ability against of blfp as a feed additive less than kg/ton against pedv after a long period of pedv inoculation. our results suggest that blfp could be a novel candidate feed additive for reducing the titer of pedv in the swine industry. effect of organic acid blend and bacillus subtilis alone or in combination on growth traits, blood biochemical and antioxidant status in broilers exposed to salmonella typhimurium challenge during the starter phase impact of porcine epidemic diarrhea on performance of growing pigs evaluation and comparison of the pathogenicity and host immune 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peptide synthetases, in non-ribosomal peptide biosynthesis in bacillus subtilis quercetin -rhamnoside reduces porcine epidemic diarrhea virus replication via independent pathway of viral induced reactive oxygen species porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines safety assessment of the antimicrobial lipopeptides synthesized by bacillus subtilis subsp. subtilis cbmdc f mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from bacillus subtilis bacillus subtilis and surfactin inhibit the transmissible gastroenteritis virus from entering the intestinal epithelial cells evaluation of genetic and developmental toxicity of surfactin c from bacillus subtilis bc publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions j-yp, y-bh, and c-yc have drafted, revised the work, and conducted the experiments; y-bh and y-hc provide specimens and substantial contributions to the conception and analysis; h-wc, y-cc, p-st, and c-rj help to interpret data; y-hc and h-wc. design the work and approve the submitted version. all authors read and approved the final manuscript. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. the animal experiment was approved by the institutional animal care and use committee (iacuc) of national taiwan university (taipei, taiwan, ntu -el- ). not applicable. the authors declare that they have no competing interests. key: cord- - jyui ca authors: lai, zheng-zong; ho, yi-jung; lu, jeng-wei title: harringtonine inhibits zika virus infection through multiple mechanisms date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: jyui ca mosquito-borne zika virus (zikv) is a flavivirus that came under intense study from to for its well-known ability to cause congenital microcephaly in fetuses and neurological guillain–barré disease in adults. substantial research on screening antiviral agents against zikv and preventing zikv infection are globally underway, but food and drug administration (fda)-approved treatments are not available yet. compounds from chinese medicinal herbs may offer an opportunity for potential therapies for anti-zikv infection. in this study, we evaluated the antiviral efficacy of harringtonine against zikv. harringtonine possessed anti-zikv properties against the binding, entry, replication, and release stage through the virus life cycle. in addition, harringtonine have strong virucidal effects in zikv and exhibited prophylaxis antiviral ability prior zikv infection. the antiviral activity also observed in the treatment against japanese encephalitis reporter virus (rp -gfp strain). overall, this study demonstrated that harringtonine would be a favorable potential candidate for the development of anti-zikv infection therapies. zika virus (zikv) is a member of flavivirus genus of the flaviviridae family and is a mosquito-borne virus with positive single-stranded rna. other flaviviruses include yellow fever virus (yfv), dengue virus (denv), west nile virus (wnv), tick-borne encephalitis viruses (tbev), and japanese encephalitis virus (jev). like these, the genome size of zikv is approximately , nucleotides in length and encodes approximately amino acids, which translate a single polyprotein. the polyprotein produces three structural proteins (capsid, pre-membrane, and envelope) and seven nonstructural proteins (ns , ns a, ns b, ns , ns a, ns b, and ns ) via viral and cellular proteolysis [ , ] . among these viral proteins, the envelope protein is responsible for viral entry and influences host attachment [ ] . however, the nonstructural proteins are related to viral rna replication and virion assembly [ ] . zikv was first discovered in rhesus macaques in ugandan forests in . the first outbreak of zikv occurred in on yap island in the western pacific ocean, followed by a large epidemic in central and south american countries in - [ ] . the spread of zikv has caused a global health concern. in addition to transmission by infected mosquitoes, zikv can also be transmitted the cytotoxicity profiles of harringtonine in non-infected vero cells were evaluated. vero cells were grown in fresh medium with raising concentrations of harringtonine in -well microplates; cytotoxicity was assessed for h using a cck- assay, which evaluated cell proliferation by measuring cellular metabolic activity. the cell viability of vero cells was approximately or % at harringtonine concentrations up to or nm after h treatment, respectively ( figure a ). to avoid drug-induced cell cytotoxicity of harringtonine treatment, this was limited to nm in subsequent antiviral experiments. to investigate the anti-zikv activity of harringtonine, we assessed the inhibition of virus infection in vero cells with moi = . under different concentrations of harringtonine for h. for this, vero cells monolayer were cultured in -well microplates overnight, infected with ffu zikv per well, and incubated with raising concentrations of harringtonine from , and nm for h. intracellular viral rna levels, protein expression levels and virus progeny in supernatants were respectively determined by rt-qpcr, western blotting and fluorescent focus assay (ffa). the dose-dependent anti-zikv activities of harringtonine were observed to decrease viral rna/protein production and progeny yield ( figure b-d) , indicating that virus propagation was suppressed. results of ifa assay also showed that harringtonine treatment inhibited zikv infection in a dose-dependent manner ( figure e ,f). taken together, our data indicated that harringtonine inhibited zikv infection by suppressing the production of zikv rna, viral protein and reducing virion yield in vitro. the experiments were carried out in triplicates, and the error bars represent standard deviation (a). vero cells were infected with zikv and were treated with different concentrations of harringtonine for h. the anti-zikv ability of harringtonine was analyzed by measuring viral rna levels (b). the cell supernatants were harvested, and virus titers were assessed by fluorecent focus assay (c). the intracellular protein expression of env was assessed by western blot (d). the severity of zikv infection was determined by ifa (e) and quantification of vero cells (f). statistical significance was analyzed from t-test compared with the zikv group: * p < . ; ** p < . ; *** p < . . scale bar: μm. to further evaluate the individual stages of the virus life cycle, a time of addition assay was performed ( figure a ). harringtonine ( nm) was administered at different stages of zikv infection with moi = . , and then the levels of zikv rna in cells, as well as virus titers in supernatants, were determined after h treatment. the zikv group and full-treatment group were, respectively, negative control and positive control. both levels of zikv rna production ( figure b ) and progeny yield ( figure c ) reduced at all processes of the virus life cycle. the strongest inhibition effect of harringtonine was observed at the post-treatment stage (approximately log inhibition of viral rna level and progeny yield), which suggested that the compound may mainly inhibit at the late stage of zikv infection. in addition, both levels of zikv rna and progeny yield were also suppressed at the co-treatment stage, which suggested that the compound may also affect at the early stage of zikv infection. in order to investigate the effect after zikv entry, vero cells were seeded and inoculated with zikv at moi = . for h absorption. then, harringtonine nm was added at , , and h after the inoculum removed. the viral rna level was detected after h incubation. based on the above data, we have determined that harringtonine effectively interferes at late stage of zikv infection ( figure d ). the viability was determined with a cell counting kit assay. the experiments were carried out in triplicates, and the error bars represent standard deviation (a). vero cells were infected with zikv and were treated with different concentrations of harringtonine for h. the anti-zikv ability of harringtonine was analyzed by measuring viral rna levels (b). the cell supernatants were harvested, and virus titers were assessed by fluorecent focus assay (c). the intracellular protein expression of env was assessed by western blot (d). the severity of zikv infection was determined by ifa (e) and quantification of vero cells (f). statistical significance was analyzed from t-test compared with the zikv group: * p < . ; ** p < . ; *** p < . . scale bar: µm. to further evaluate the individual stages of the virus life cycle, a time of addition assay was performed ( figure a ). harringtonine ( nm) was administered at different stages of zikv infection with moi = . , and then the levels of zikv rna in cells, as well as virus titers in supernatants, were determined after h treatment. the zikv group and full-treatment group were, respectively, negative control and positive control. both levels of zikv rna production ( figure b ) and progeny yield ( figure c ) reduced at all processes of the virus life cycle. the strongest inhibition effect of harringtonine was observed at the post-treatment stage (approximately log inhibition of viral rna level and progeny yield), which suggested that the compound may mainly inhibit at the late stage of zikv infection. in addition, both levels of zikv rna and progeny yield were also suppressed at the co-treatment stage, which suggested that the compound may also affect at the early stage of zikv infection. in order to investigate the effect after zikv entry, vero cells were seeded and inoculated with zikv at moi = . for h absorption. then, harringtonine nm was added at , , and h after the inoculum removed. the viral rna level was detected after h incubation. based on the above data, we have determined that harringtonine effectively interferes at late stage of zikv infection ( figure d ). . zikv rna levels were determined using rt-qpcr at different infection processes (b). virus titers in supernatants were determined using fluorescent focus assay (ffa) (c). the nm of harringtonine was added at , , and hpi (hour post infection). at hpi, the viral rna level was analyzed by rt-qpcr (d). statistical significance was analyzed from t-test compared with the zikv group: * p < . ; ** p < . ; *** p < . . statistical significance was analyzed from t-test compared with the zikv group: * p < . ; ** p < . ; *** p < . . our previous studies have shown that virus could bind onto cells, but could not enter into the cells at • c. thus, °c utilized to verify the effect of harringtonine on zikv absorption and then removed the inoculum and washed which could focus on the effect of virus binding. to further clarify the inhibitory process of harringtonine at the co-treatment stage, temperature difference-based binding assay and entry assay, replication, and release assay were conducted. the nm harringtonine was added during zikv infection (moi = . ) at °c, then the treated cells were washed, and fresh medium was added at °c. after h, rna and virus titer were detected and used to verify the effect of viral binding. furthermore, the cells were infected with zikv (moi = . ) at • c for h incubation, then the inoculum was removed, washed to replace with nm harringtonine, and incubated at • c for another hour. subsequently, nm harringtonine was replaced with fresh medium, which was used to investigate the effect of viral entry. the results demonstrated that harringtonine could inhibit zikv infection with moi = . through virus binding ( figure a ,b) and virus entry processes ( figure c,d) . besides, the cells were infected with zikv (moi = . ) at • c for h, and then incubated at • c for another hour. when zikv entered into the cell, the specified concentration of harringtonine was added. the rna level was used to verify whether harringtonine affected virus rna replication, and the viral progeny yield was used to further determine whether harringtonine influenced virus release. these results indicated that harringtonine also reduced the viral rna replication and virion release ( figure e ,f) by rt-qpcr and ffu assay. moreover, we also assessed whether harringtonine affects zikv stability. the zikv supernatant was added with the specified concentration of harringtonine for h incubation to verify the virucidal ability. then, the zikv supernatant was progressed -fold serial-diluted and ffa was applied to determine the reduction of virus stability. therefore, the above experiments could verify the effects of different stages. the results of harringtonine were to inhibit zikv stability at a concentration of and nm ( figure g ). we produced a timeline of time of binding, entry, replication, and release assays ( figure h ). interestingly, the antiviral effect of harringtonine was also observed even at the pre-treatment stage, when cells were pre-treated with harringtonine for h before virus infection (figure a,b) . the result of the analysis of molecular docking was used to measure the likelihood of harringtonine binding to the zikv envelope proteins, the highest patchdock score was ( figure a,b) . overall, the above evidence indicated that harringtonine possessed antiviral activities by not only disrupting viral replication, release but also blocking viral binding, entry, as well as exhibiting prophylactic effect before zikv infection. binding, entry, replication and release assays. the red line refers to the zikv absorption period and dotted blue line refers to the harringtonine administration period (h). statistical significance was analyzed from t-test compared with the zikv group: * p < . ; ** p < . . to further assess the antiviral activity of harringtonine against other virus, the japanese encephalitis reporter virus (rp -gfp strain) with moi = . was used. briefly, vero cells were seeded in -microplates overnight. cells were then infected with ffu rp -gfp virus in the presence of harringtonine at different concentrations for h. intracellular viral rna levels were determined using rt-qpcr. the inhibitory effect on viral infection was evaluated by observing virus fluorescent protein expression under an inverted fluorescence microscope. results showed that to nm harringtonine exhibited a dose-dependent anti-rp -gfp activity ( figure a-c) . consequently, harringtonine exhibited an antiviral potential to be used in the treatment of jev infection. to further assess the antiviral activity of harringtonine against other virus, the japanese encephalitis reporter virus (rp -gfp strain) with moi = . was used. briefly, vero cells were seeded in -microplates overnight. cells were then infected with ffu rp -gfp virus in the presence of harringtonine at different concentrations for h. intracellular viral rna levels were determined using rt-qpcr. the inhibitory effect on viral infection was evaluated by observing virus fluorescent protein expression under an inverted fluorescence microscope. results showed that to nm harringtonine exhibited a dose-dependent anti-rp -gfp activity ( figure a-c) . consequently, harringtonine exhibited an antiviral potential to be used in the treatment of jev infection. zikv infection in neonates with congenital microcephaly born from zikv-infected pregnant women was identified as an emerging health issue in brazil from to . to prevent the severe consequences of zikv infections and reduce zikv-induced neurological defects, an effective anti-zikv agent is required. compounds isolated from natural plants have been widely evaluated in many antiviral studies [ , ] . previous research on the natural cephalotaxus alkaloids harringtonine, homoharringtonine, and cephalotaxine majorly showed antitumor effects, such as antileukemic activity [ , , ] . cephalotaxus alkaloid antitumor effects were suggested via their inhibitory effects on protein synthesis and partly on dna synthesis [ , ] . however, further research on cephalotaxus alkaloids demonstrated antiviral effects against hepatitis b virus (hbv), bovine viral diarrhea virus (bvdv), chikv, vzv, foot and mouth disease virus (fmd), vesicular stomatitis virus (vsv), newcastle disease virus (ndv), and sars-cov- [ , , [ ] [ ] [ ] [ ] [ ] [ ] . in this study, the drug-induced cytotoxicity of harringtonine was first assessed in vero cells, the vero cells have been previously reported to be highly permissive for zikv growth and replication [ ] . the concentration of harringtonine used in this anti-zikv study was no more than nm to avoid drug-induced cytotoxicity and kept the cell viability of vero cells remained above % ( figure a ). this concentration of harringtonine was much lower than that used in the treatment of chikv with µm harringtonine in bhk cells [ ] . this distinction may be due to the different cell lines and experimental approaches used. a time of addition experiment was conducted to determine which stage of zikv life cycle was disrupted. harringtonine treatments exhibited anti-zikv effects on all virus life stages including co-treatment, post-treatment, and even pre-treatment ( figure ) . noticeably, post-treatment of harringtonine showed that the most potent inhibitory effects on intracellular viral rna level and viral progeny yield in supernatants. harringtonine also exhibited a prophylactical antiviral activity before zikv infection in vitro, suggesting that harringtonine probably entered and was retained in the cells and then exerted inhibitory effects. a previous study demonstrated that harringtonine decreased in chikv rna synthesis and protein production and also reduced viral progeny. furthermore, harringtonine also presented the prophylactical antiviral activity in chikv infection. [ ] . denv-infected cells revealed the increase of subpolysomal mrnas which might correlate to the repression of translation at the initiation stage [ ] . harringtonine, through a block following s subunit joining, could inhibit the initiation of translation, and be used to verify if denv infection could affect the translation elongation. denv infection could disrupt the host cell translation at the starting stage, but does not change the translation elongation [ ] . other studies reported that harringtonine inhibited protein synthesis by blocking poly(u)-directed polyphenylalanine synthesis and peptide bond formation [ ] , and interfered with large ribosome subunit [ ] . therefore, the decrease of viral protein expression might also relate to down-regulating protein synthesis. previous evidences indicated that harringtonine was an inhibitor of protein synthesis, which is most likely to inhibit the large ribosomal unit of eukaryotes, thereby inhibiting the translation of non-structural or structural proteins [ , ] . the above evidence implies that the antiviral activities of harringtonine might occur on host factors. harringtonine was effective against sindbis virus (sinv) and chikv and exhibits a dose-dependent inhibitory effect, but it is not effective in inhibiting the growth of encephalomyocarditis virus (emcv). therefore, the antiviral effect of harringtonine may be limited to related viruses transmitted by mosquitoes [ , ] . to further clarify the inhibitory activities of harringtonine that occurred at the co-treatment stage, binding assay and entry assay were performed. the results indicated that harringtonine could block both viral binding and entry into host cells based on the decrease in viral rna production ( figure a ,c) and virion progeny ( figure b,d) . harringtonine also revealed the virucidal ability which could destroy the virion stability ( figure g ). through molecular docking, harringtonine was predicted the binding affinity of the envelope of zikv, which might be the reason of harringtonine blocking the early stage of zikv infection and affecting the virion stability. compared to our previous study of cephalotaxine, harringtonine obviously possessed more complex mechanisms against zikv infection than cephalotaxine, in blocking virus binding, entry, stability and possessing prophylactical antiviral ability. the results mean that harringtonine treatment could be used in more complicated medical conditions against zikv infection. both of the in vivo and clinical treatments, these results proved the safety or lower drug toxicity of harringtonine [ ] [ ] [ ] . in our previous study, we also demonstrated that cephalotaxine exhibited anti-zikv and denv activity in vitro. although both harringtonine and cephalotaxine belong to cephalosporin isolates, they still have different anti-zikv abilities. first of all, the ec and cc of harringtonine are . nm and > µm, while the ec and cc of cephalotaxine are µm and > µm. the selectivity index (si) of harringtonine is . , while the si of cephalotaxine is . . second, both harringtonine and cephalotaxine have inhibitory effects after virus entry, because they have inhibitory effects in post-infection treatment. however, harringtonine revealed more effects on zikv binding and entry, whereas cephalotaxine did not observe the same effect. third, the virucidal assay showed that µm cephalotaxine reduced the infection ability of zikv by about %, but did not show more effects at a concentration of µm (data not shown) [ ] . however, the harringtonine has a better inhibitory effect ( . % at nm and . % at nm) in the virucidal assay, and it is dose-dependent. in this study, harringtonine possessed the ability against zikv infection during virus binding, entry, and virucidal assay. furthermore, the molecular docking showed that harringtonine could bind with zikv envelope protein ( figure ). the envelope protein was the most important structural protein of zikv and responded for virus binding and entry. based on the above new findings, harringtonine is more conducive to becoming a new candidate drug against zikv infection than cephalotaxine. despite these compounds sharing similar structures, their multiple biological and pharmacological activities may vary with different side chains [ ] . in this study, the anti-jev effects of harringtonine was also investigated and demonstrated that harringtonine possessed dose-dependent antiviral activities ( figure a -c). in conclusion, harringtonine was proved to suppress zikv and jev infection, and all of those evidence indicated that cephalotaxus alkaloids might possess the broad-spectrum anti-viral effects in flaviviruses through multiple mechanisms. african green monkey kidney cells (vero; atcc, ccl- ) was used in this study, as it is more permissive to zikv (pravabc ; genbank sequence accession: ku ) replication; vero cells were cultured in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs) and antibiotics under a % co incubator at • c. harringtonine was purchased from chemfaces (catalog number: cfn ), dissolved in % dimethyl sulfoxide (dmso) as a stock of mm, and stored at − • c until use. green fluorescence protein-expressing japanese encephalitis reporter virus (rp -gfp strain) was kindly given to us by dr. lin ren-jye. the propagation and titration of zikv were performed using vero cells. virus titer was determined using the fluorescent focus assay (ffa). the propagation and titration of jev were conducted using c / mosquito cells (c / ; atcc, crl- ) and vero cells, respectively. cytotoxicity of harringtonine was determined using the cell counting kit (cck- , dojindo laboratories, kumamoto, japan). increasing concentrations of harringtonine with fresh medium were added to cells in -well microplates in triplicates and incubated at • c in a % co incubator. after a or h incubation period, the medium was replaced with µl of fresh medium containing µl of cck- reagent for h. the absorbance at nm was measured using an elisa reader (synergy ht, biotek, winooski, vt, usa). the cell viability values for treated cells were normalized with those of untreated cells. total rna including viral genomic rna was extracted from infected cells using total rna reagent (bioman, tri ), after that rna levels were measured using the one-step × rt-qpcr mix sybr green kit (bioman; catalog number: qrp ). rt-qpcr was performed on a roche lightcycler (roche applied science, indianapolis, in) at the following conditions: • c, min; • c, min; ( • c, s; • c s; • c s) for cycles. the primers used to detect zikv, jev and β-actin (internal control) were as follows: zikv: forward primer '-ttggtcatgatactgctgatgc- and reverse primer '-ccttccacaaagtccctattgc- ', jev: forward primer '-tccgtcaccatgccagtctt- ' and reverse primer '-gaggatgattctgtaagtatctaggtatagagccc- ', and b-actin: forward primer '-aggcaccagggcgtgat- ' and reverse primer '-gcccacataggaatccttc tgac- ' [ ] . data were analyzed using the -ct method. viral titers were performed by using ffa. briefly, the virus solution was serially diluted and added to monolayer vero cells. the medium was discarded after a h incubation time, and cells were overlaid under semisolid dmem containing . % methylcellulose for h. subsequently, assay of immunofluorescence was conducted and fluorescent viral foci were counted under an inverted fluorescence microscope (whited). results of viral titers were expressed as fluorescent focus units per ml (ffu/ml). cells cultured in -well plates were fixed with % formaldehyde at room temperature for h and were then permeabilized with an equal ratio of chilled methanol and acetone ( : ) for min. cells were then washed three times with phosphate-buffered saline (pbs) and blocked with % skimmed milk for h and were stained with anti-flavivirus envelope protein g primary antibody ( : dilution; produced in-house) for h. subsequently, cells were washed three times with pbs again, and alexa fluor -conjugated goat anti-mouse igg secondary antibody ( : dilution; jackson immunoresearch laboratories, inc.; west grove, pa, usa) was added at • c for another h incubation. stained cells were visualized using an inverted fluorescence microscope (olympus ckx , olympus, japan), as previously reported [ ] . total cell lysates were harvested by adding ripa lysis buffer supplemented with protease inhibitors. anti-flavivirus envelope protein g antibodies ( : dilution; produced in-house) and rabbit anti-β-actin polyclonal antibodies ( : dilution; finetest, wuhan, china, catalog number: fnab ) were used as primary antibodies. anti-mouse or anti-human horseradish peroxidase-conjugated antibodies were used as secondary antibodies. blots were developed by adding enhanced chemiluminescence (ecl) reagent. vero cell monolayers were cultured overnight in -well microplates. drug-containing medium ( nm of harringtonine) was added at different time points relative to the h period of cell infection with approximately ffu of zikv (moi = . ). for the pre-treatment group (pre), harringtonine was added h before the virus infection. for the co-treatment group (co), harringtonine was added at the beginning of the virus infection. for the post-treatment group (po), harringtonine was added after the h period of infection. for the full-duration treatment group (full), harringtonine was added throughout the infection. cells were washed twice with pbs in each stage. after h incubation, cells and supernatants in all groups were harvested. the levels of intracellular viral rna and virus titers from supernatants were determined by rt-qpcr and ffa, respectively, as previously described [ , , ] . for binding assay, zikv (moi = . ) were inoculated onto vero cell monolayers in culture medium with or without nm of harringtonine for h at • c. cells were then washed twice with pbs, and fresh medium was added for h incubation at • c in a % co incubator. the levels of intracellular viral rna and virus titers in supernatants were determined by rt-qpcr and ffa, respectively. for entry assay, fresh medium containing zikv (moi = . ) was added to vero cells at • c for h. cells were then washed twice with pbs, and fresh medium with or without nm of harringtonine was added for another h incubation at • c in a % co incubator. thereafter, cells were washed twice with pbs again before being added to fresh medium. after a h incubation period, the levels of intracellular viral rna and virus titers in supernatants were determined by rt-qpcr and ffa, respectively [ , ] . the cells were infected with zikv (moi = . ) at • c for h, and then incubated at • c for another hour. when zikv entered into the cell and added the specified concentration of harringtonine. after day incubation, the cells lysate were collected for viral rna replication assay by rt-qpcr and the supernatant was assessed by the ffu assay to determine the viral release [ , ] . zikv ( × ffu) was respectively mixed with harringtonine at , , , and nm at • c for h; virus titers were then determined by ffa [ ] . the zikv envelope proteins ( ire) crystal structure was obtained from the protein data bank (pdb). three-dimensional ligand structures of (chemspider id: ) was obtained from the chemspider database and converted into the pdb format using pymol software, as previously reported [ ] . patchdock was used in this project to conduct molecular docking analyses. finally, the conformations were then ranked according to docking scores [ ] . the data obtained from this study were statistically analyzed in triplicate and using graphpad prism . software (graphpad software inc., san diego, ca, usa), and values were expressed as mean ± standard deviation. the statistical analyses of data were calculated using a two-tailed student's t-test, where a p-value < . was considered significant. full-length sequencing and genomic characterization of bagaza, kedougou, and zika viruses zika virus: history, emergence, biology, and prospects for control structures of the zika virus envelope protein and its complex with a flavivirus broadly protective antibody pathogenesis of flavivirus infections: using and abusing the host cell zika virus outbreak on yap island, federated states of micronesia potential sexual transmission of zika virus risk of zika virus transmission by blood donations in brazil zika virus infection and the eye microcephaly and zika virus infection study links zika virus to guillain-barre syndrome structures of harringtonine, isoharringtonine, and homoharringtonine anti-varicella-zoster virus activity of cephalotaxine esters in vitro inhibition of chikungunya virus replication by harringtonine, a novel antiviral that suppresses viral protein expression identification of inhibitory compounds against singapore grouper iridovirus infection by cell viability-based screening assay and droplet digital pcr comparative in vitro antitumor activity of homoharringtonine and harringtonine against clonogenic human tumor cells cephalotaxine inhibits zika infection by impeding viral replication and stability herb-target interaction network analysis helps to disclose molecular mechanism of traditional chinese medicine effect of feeding chinese herb medicine ageratum-liquid on intestinal bacterial translocations induced by h n aiv in mice new natural products in cancer chemotherapy molecular modes of action of cephalotaxine and homoharringtonine from the coniferous tree cephalotaxus hainanensis in human tumor cell lines inhibition of translation in eukaryotic systems by harringtonine harringtonine, an inhibitor of initiation of protein biosynthesis in vitro" models of hepatitis b virus (hbv) and bovine viral diarrhoea virus (bvdv) replication inhibitory effects of homoharringtonine on foot and mouth disease virus in vitro the natural compound homoharringtonine presents broad antiviral activity in vitro and in vivo shedding light on the effect of natural anti-herpesvirus alkaloids on sars-cov- : a treatment option for covid- remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov- replication in vitro comparative analysis of different cell systems for zika virus (zikv) propagation and evaluation of anti-zikv compounds in vitro flavivirus infection uncouples translation suppression from cellular stress responses u determines the species specificity of the a-site cleft antibiotics: the structures of tiamulin, homoharringtonine, and bruceantin bound to the ribosome specificity of protein synthesis inhibitors in the inhibition of encephalomyocarditis virus replication uptake, initial effects, and chemotherapeutic efficacy of harringtonine in murine leukemic cells sensitive and resistant to vincristine and other chemotherapeutic agents antitumor activities of harringtonine and homoharringtonine, cephalotaxus alkaloids which are active principles from plant by intraperitoneal and oral administration long survival in an elderly patient with acute myeloid leukaemia after treatment with harringtonine palmatine inhibits zika virus infection by disrupting virus binding, entry, and stability antiviral activities of niclosamide and nitazoxanide against chikungunya virus entry and transmission micafungin is a novel anti-viral agent of chikungunya virus through multiple mechanisms suramin inhibits chikungunya virus entry and transmission patchdock and symmdock: servers for rigid and symmetric docking we wish to thank yen-mei lee and hsin-hsuen shen for their experimental assistance. the authors declare no conflict of interest.molecules , , key: cord- -fbddxbhd authors: ko, meehyun; jeon, sangeun; ryu, wang‐shick; kim, seungtaek title: comparative analysis of antiviral efficacy of fda‐approved drugs against sars‐cov‐ in human lung cells date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: fbddxbhd drug repositioning represents an effective way to control the current covid‐ pandemic. previously, we identified fda‐approved drugs which exhibited substantial antiviral effect against severe acute respiratory syndrome coronavirus in vero cells. since antiviral efficacy could be altered in different cell lines, we developed an antiviral screening assay with human lung cells, which is more appropriate than vero cell. the comparative analysis of antiviral activities revealed that nafamostat is the most potent drug in human lung cells (ic( ) = . µm). coronavirus disease (covid- ) is an emerging infectious disease caused by a coronavirus. the causative virus was named as severe acute respiratory syndrome coronavirus (sars-cov- ) because it is very similar to sars-cov ( . %) and this virus belongs to the betacoronavirus genus within the coronaviridae family. both sars-cov and middle east respiratory syndrome coronavirus (mers-cov) also belong to the same betacoronavirus genus. neither vaccine nor therapeutic has been developed for sarsand mers-cov and the current standard of care for the patients with covid- is just supportive care. however, numerous clinical trials are ongoing globally with food and drug administration (fda)approved drugs as drug repositioning programs (https://www.covidtrials.org/). among these drugs, (hydroxy)chloroquine, lopinavir/ ritonavir, and remdesivir are those that are the most frequently being tested worldwide due to the well-known in vitro antiviral effects on both mers-and sars-cov and even on sars-cov- . previously, we identified a total of potential antiviral drug candidates from fda-approved drugs using vero cells. since antiviral efficacy could be altered in different cell lines, we developed a new image-based antiviral screening assay with calu- cells, a well-known human lung cell line, and compared the antiviral efficacy of the antiviral candidates in between vero and calu- cells. calu- used in this study is a clonal isolate, which shows higher growth rate compared with the parental calu- obtained from the american type culture collection (atcchtb- ). calu- was maintained at °c with % co in eagle's minimum essential medium (emem, atcc), supplemented with % heat-inactivated fetal bovine serum (fbs) and x antibiotic-antimycotic solution (gibco). sars-cov- (βcov/kor/kcdc / ) was provided by korea centers for disease control and prevention (kcdc), and was propagated in vero cells. viral titers were determined by plaque assays in vero cells. all experiments using sars-cov- were performed at institut pasteur korea in compliance with the guidelines of the knih, using enhanced biosafety level (bsl- ) containment procedures in laboratories approved for use by the kcdc. in our previous drug repositioning study, we identified a total of potential antiviral drug candidates from fda-approved drugs. (table ) , the ic of remdesivir rather decreased by folds compared with that with vero cells, perhaps due to the low metabolic capacity or prodrug activation rate in vero cells ( table ). these discrepancies might in part account for the different outcomes from numerous clinical trials using chloroquine, lopinavir, and remdesivir. so far, the treatment with (hydroxy) chloroquine or lopinavir/ritonavir did not show any promising results concerning the covid- treatment - ; however remdesivir seems to be effective for treatment of patients with covid- in certain clinical settings. interestingly, the ic values of most drugs in our study increased in varying degrees in calu- cells (figures a,c) (tables and ). only six drugs showed decreases in ic ( figure b ) ( table ) : nafamostat mesylate, camostat mesylate, remdesivir, hydroxyprogesterone caproate, digitoxin, and cyclosporine. although nafamostat mesylate and camostat mesylate were not selected as potent antiviral drug candidates in our earlier study, we compared the antiviral efficacy of these drugs at this time in between vero and calu- cells following the discovery that tmprss , a host protease necessary for priming viral spike glycoprotein, could be a target for covid- antiviral development. the discrepancy in ic was specifically remarkable with nafamostat mesylate; the ic decreased by approximately folds when the drug was used in the sars-cov- -infected calu- cells perhaps due to the dominant role of tmprss -dependent viral entry in the calu- human lung epithelial cells. , in addition, the ic of nafamostat mesylate was exceptionally low ( . µm), which indicates that nafamostat mesylate is approximately -fold more potent than remdesivir in calu- cells. it became more apparent that blood clotting is one of the complicating manifestations in patients with covid- , , and nafamostat mesylate may play dual roles not only as an antiviral to block viral entry but also as an anticoagulant to remove blood clots frequently associated with acute respiratory distress syndrome. a recent case report on the treatment of three patients with covid- with nafamostat and other in vitro studies , corroborated our findings. however, nafamostat has a short half-life in the serum, thus requires continuous intravenous injection, which disables convenient administration for a large group of patients. solving this disadvantage will increase the accessibility of more covid- patients to nafamostat treatment. in summary, we compared antiviral efficacy of the potential antiviral drug candidates against sars-cov- in between vero and calu- cells and found that nafamostat mesylate is the most potent antiviral drug candidate in vitro. importantly, nafamostat mesylate has been approved for human use in japan and korea for over a decade, thus it can be readily repurposed for covid- following phase ii-iii clinical trials. currently, a few clinical trials have been registered (https://clinicaltrials.gov/). according to our results, although in vivo animal models are preferred experimental systems for evaluating antiviral efficacy, in vitro testing using human lung cells is the authors declare that there are no conflict of interests. a pneumonia outbreak associated with a new coronavirus of probable bat origin the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro identification of antiviral drug candidates against sars-cov- from fda-approved drugs cultured human airway epithelial cells (calu- ): a model of human respiratory function, structure, and inflammatory responses remdesivir potently inhibits sars-cov- in human lung cells and chimeric sars-cov expressing the sars-cov- rna polymerase in mice observational study of hydroxychloroquine in hospitalized patients with covid- effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus (sars-cov- ) infection a trial of lopinavir-ritonavir in adults hospitalized with severe covid- remdesivir for the treatment of covid- -preliminary report sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor identification of nafamostat as a potent inhibitor of middle east respiratory syndrome coronavirus s protein-mediated membrane fusion using the split-protein-based cellcell fusion assay simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry incidence of thrombotic complications in critically ill icu patients with covid- pulmonary embolism in covid- patients: awareness of an increased prevalence three cases of treatment with nafamostat in elderly patients with covid- pneumonia who need oxygen therapy nafamostat mesylate blocks activation of sars-cov- : new treatment option for covid- the anticoagulant nafamostat potently inhibits sars-cov- s protein-mediated fusion in a cell fusion assay system and viral infection in vitro in a cell-typedependent manner comparative analysis of antiviral efficacy of fda-approved drugs against sars-cov- in human lung cells conception: wsr and sk. study design: mk, wsr, and sk. participation in experiments and data collection: mk and sj. data acquisition, writing manuscript, and statistical analysis: mk and sk. all authors reviewed the manuscript and approved the final version. the data that support the findings of this study are available from the corresponding author upon reasonable request. seungtaek kim http://orcid.org/ - - - key: cord- -t ykkekl authors: stone, e. taylor; geerling, elizabeth; steffen, tara l.; hassert, mariah; dickson, alexandria; spencer, jacqueline f.; toth, karoly; dipaolo, richard j.; brien, james d.; pinto, amelia k. title: characterization of cells susceptible to sars-cov- and methods for detection of neutralizing antibody by focus forming assay date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: t ykkekl the sars-cov- outbreak and subsequent covid- pandemic have highlighted the urgent need to determine what cells are susceptible to infection and for assays to detect and quantify sars-cov- . furthermore, the ongoing efforts for vaccine development have necessitated the development of rapid, high-throughput methods of quantifying infectious sars-cov- , as well as the ability to screen human polyclonal sera samples for neutralizing antibodies against sars-cov- . to this end, our lab has adapted focus forming assays for sars-cov- using vero ccl- cells, referred to in this text as vero who. using the focus forming assay as the basis for screening cell susceptibility and to develop a focus reduction neutralization test. we have shown that this assay is a sensitive tool for determining sars-cov- neutralizing antibody titer in human, non-human primate, and mouse polyclonal sera following sars-cov- exposure. additionally, we describe the viral growth kinetics of sars-cov- in a variety of different immortalized cell lines and demonstrate via human ace and viral spike protein expression that these cell lines can support viral entry and replication. which human cell types may be more permissive to sars-cov- infection, with particular uncertainty including if higher ace expression coincides with increased risk for heightened infection. furthermore, it is unclear which cell types may be suitable for the successful evaluation of antiviral and therapeutic drugs for in vitro screening before in vivo evaluation. further characterization of permissible cell types is necessary to improve our understanding of sars- cov- pathogenesis and to develop therapeutic strategies for the treatment of it has also been noted that following infection with sars-cov- , people typically seroconvert days following symptom onset, and there is increasing evidence suggesting that development of a neutralizing antibody response is a correlate of protection in patients recovered from . the covid- pandemic has highlighted the necessity for testing for neutralizing antibodies. as sars-cov- infection can have a range of manifestations from asymptomatic to fatal multiple organ failure [ ] [ ] [ ] [ ] [ ] , antibody testing and serological surveys are a critical tool for determining prior infection status and seroprevalence in a population . it is also the goal of many candidate sars-cov- vaccines to induce neutralizing antibodies targeting the viral spike protein, the major antigenic determinant of sars-cov- [ ] . for understanding the antibody response, assays that measure neutralizing antibody titer are considered the gold standard. one such tool for evaluating neutralizing antibody response is a plaque/focus neutralization reduction test (prnt/frnt), which evaluates the ability of polyclonal sera samples to prevent or reduce infection of a cell monolayer in vitro. previously, for sars- cov- , only prnt assays-which rely on the ability of virus to lyse infected cells and thus can take - hours to develop-have been used in the assessment of the neutralizing antibody response. it is not well known whether an frnt-which uses an immunostaining protocol to detect virus and does not depend on cell lysis, and thus is often more rapid-is amenable for detection of sars-cov- neutralizing antibody. in this study, we describe the growth kinetics of sars-cov- in multiple cell types and the methods our laboratory has used to optimize a sars-cov- focus forming assay (ffa) to improve sensitivity and specific detection. by characterizing the growth kinetics of sars-cov- on a variety of immortalized and primary cell lines, we have demonstrated which of these cell lines is susceptible to infection by sars-cov- .we also demonstrate that the ffa can be adapted to measure the neutralization capacity of polyclonal sera in an frnt. this high throughput frnt assay can be applied to sera from both animal models of sars-cov- infection, as well as human sars-cov- infected patients, and can serve as a useful assay for describing the kinetics of the neutralizing antibody response to sars-cov- . additionally, we have compared the expression of ace and sars-cov- spike on these cell lines to determine how spike expression correlates with susceptibility. the tools developed in this study have practical applications in both the basic science and translational approaches that will be critical in the ongoing effort to slow the covid- pandemic. based on our previous work optimizing the ffa for wnv, [ ] we proceeded to adapt the ffa for the detection of infectious sars-cov- . along with spot number, the spot size and border definition provide valuable information on possible differences in viral strains. as we have observed that the foci morphology, as well as spot number, can vary dramatically under different growth conditions, we sought to test different growth conditions and cell lines to determine the optimal conditions for sars-cov- viral titration. this goal was guided by previous studies that have suggested that the use of vero cells from varying origins can impact viral titer [ , ] . using both vero ccl- (atcc® ccl- ™, referred to in this text as vero who) and vero e (vero , atcc® crl- ™) cell lines, we determined if differences in foci number or size occurred to decide if one cell line was superior for titration by ffa (figure a- c) . although many laboratories utilize vero e cells for viral titer measurements of sars-cov- [ , ] , in our laboratory, vero e cells typically resulted in about two-fold lower foci formation relative to vero who cells (figure a-c) . figure a is a representative image of an ffa showing the viral titration on both the vero e and whos for both ~ ffu and ~ ffu when identical numbers of cells are seeded per well. we noted that at identical higher dilutions of sars-cov- virus stocks, vero who cells develop ~ individual foci per well, whereas vero e cells develop ~ foci per well ( figure a) . the same pattern was observed at lower dilutions of virus, with ~ foci formation on vero who cells yielding only ~ foci on vero e cells ( figure a ) the quantification of this difference in the vero who and vero e cell type is shown in figure b. interestingly, when we compared this observation to the genome copy number by we noted that vero e cells tended to produce significantly more virus across all , , and - hour timepoints (p = . ) ( figure c) . it is possible that the discrepancy between the ffa and qrt-pcr data could be due to vero who cells producing fewer genome copies yet more infectious virus than e cells, while e cells sustain more viral replication but yield less infectious virus. while we did not see any differences in foci morphology between the two vero cell lines we used, the significant difference in the number of foci observed between vero e and vero who (p < . ) suggests that vero who cells record higher viral titers than the vero e cells for sars-cov- ( figure b) . in order for sars-cov- to form distinct foci, it is critical to plate the optimal cell density. we examined the impact of cell density on foci formation for both vero who and vero e cells by plating identical dilutions of sars-cov- virus stocks on -well plates seeded with differing numbers of who or e cells ( × , . × or × cells/well) one day prior to infection of the cell monolayer. at these concentrations, on the day of infection, × , . × and × cells/well resulted in monolayers that were , and percent confluent respectively for both cell lines tested. figure d is a representative image of the focus forming assay showing the foci formations arising from different cell concentrations plated for both the vero e and whos. the quantification of the spot counts for vero who cells is shown in figure e . the quantification of the spot counts for vero e cells is shown in figure f . for both the vero whos and e cells, we observed a significant increase in the number of foci formed when either . × (e p = . , who p = . ) or × (e p = . , who p = . ) were seeded per well compared to × . however, there was no significant increase in foci formation when increasing the cell density from . × cells/well to × cells/well. thus, while the vero e cells fostered lower foci numbers at each confluency as compared to the who cells, viral titers were not significantly different at . × cells/well or × cells/well irrespective of whether the vero who (figure e ) or vero e ( figure f ) cell line was seeded for the assay. we have previously tested higher cell densities for ffas and have noted that cell concentrations higher than × cells/well results in an overly confluent monolayer with more cells than can adhere to the wells, leading to highly variable titer information (data not shown). the results of these studies suggest that vero who cells plated at either . × or × cells/well was optimal for the viral ffa. like plaque assays, the incubation time for the ffa is highly dependent on the viral replication cycle within the cells and the time required for infectious progeny to be released and spread to neighboring cells. whileunlike traditional plaque assaysthe ffa is not dependent on viral lysis of infected cells, the development of visible spots is dependent on the time it takes for viral protein production to occur and for infectious virus to spread to neighboring susceptible cells. to determine the optimal time frame for infection of sars-cov- on a vero who cell monolayer to form individual foci, we tested a variety of incubation times. in order to optimize these conditions, identical dilutions of sars-cov- virus stocks were added to infect vero who cells seeded at a density of . × cells/well, and incubated for , , , or hours post infection. figure g shows representative images of identical dilutions of sars-cov- ffas developed after , , or hour incubation times, respectively. the mean spot size of foci at each timepoint is quantified in figure h . the mean spot count per well at each time point is quantified in figure i . altering the incubation time had the most dramatic impact on mean foci size amid all other parameters tested. while there were no significant differences in the size of foci formed between hours post infection (hpi) and hpi (p = . ), we did observe the formation of significantly larger foci between the and hpi timepoints (p = . ) , as well as the and hpi timepoints (p = . ) ( figure h) . interestingly, at the same virus dilution, we found that there were fewer spots between hpi (mean spot number of ) relative to the hpi time point (p = . , mean spot number of . ) but not the hpi time point (p = . , mean spot value . ). similarly, we found that there were no significant differences in the number of foci formed between and hpi (p = . ) ( figure i ). however, we noted that this difference is within one standard deviation of the mean number of spots across all wells and was insignificant when determining titers. from this result we concluded that incubation times greater than hours resulted in a slight but significant increase in spot number, while assays incubated for up to did not alter the spot number but did increase the spot size. this increase in spot size but not number between and hours is a highly useful for the testing of anti-viral compounds which may require longer incubation times. in addition, larger spot size makes this assay more universally useful since laboratories without an automated machine can manually count spots, where the large size will improve readability of the assay. the ffa relies on an immunostaining protocol of an infected cell monolayer in order to quantify infectious virus titer and is therefore dependent upon sars-cov- -specific antibody binding. for this purpose, polyclonal guinea pig sera (bei: nr- ) raised against sars-cov- produces reproducible staining with minimal background. however, we have also used human as efforts to understand replication and transmission of sars-cov- are underway and vaccine development moves forward, more information regarding permissive cell types for sars- cov- infection and replication is needed. to determine the permissivity of several different cell types to infection with sars-cov- , we generated multistep growth curves for human, non- human primate, murine, hamster, and gastric adenocarcinoma cell lines. each of these cell lines was infected at a moi = . and cells or cell supernatants were collected aseptically, and total rna was isolated. cellular rna was normalized to an internal rnasep control for human and non-human primate cells and gapdh for murine cells. genome equivalents were determined using the applied biosystems taqman gene expression assay protocol for sars-cov- previously described [ ] . to identify susceptible cell lines, we first assessed genome copy number in non-human primate african green monkey kidney epithelial cells (vero who, vero e ) as well as human hepatocytes (huh . , huh ) and lung epithelial cells (a , calu- ). figure a shows the sars-cov- genome copy number for whole cells for human and non-human primate cell lines. figure b shows the sars-cov- genome copy number for cell culture supernatants for human and non-human primate cell lines. in each cell line aside from vero who and huh , genome equivalents within the cell peaked at hpi and remained relatively constant for the duration of the experiment. sars-cov- genome equivalents in vero who and huh cells peaked at hpi and remained relatively constant for the duration of the experiment. the vero e cell line reached the highest titer at . × copies/µl at hpi, with the huh . and calu- cell lines reaching . × copies/µl and . × copies/µl, at the same time point, respectively. vero who and huh cell lines reached the highest titer, . × copies/µl and . × copies/µl, respectively, at hpi. the a cells, although susceptible to infection, appeared to support little sars-cov- replication, reaching only copies/µl at hpi. we also measured genome copy number in the cell culture supernatant for all of the cell lines described ( figure b ) for each cell line, viral rna in the supernatant peaked at later timepoints, either hpi (vero who, . × copies/µl; vero e , . × copies/µl; calu- , . × copies/µl) or hpi (a , . copies/µl; huh , copies/µl; huh . , . × copies/µl). of these cell lines, the vero who cells had the highest titers in the supernatant, while vero e , huh . and calu- cells were comparable in terms of titer. as expected, a cells that did not contain high titers of cell-associated virus also did not contain high titers in the supernatant. interestingly, while huh cells support relatively high titers of cell-associated virus, they do not appear to yield high titers in the supernatant. these results suggest that vero e cells are most permissible for sars-cov- replication among all tested cell types and would be the ideal choice for propagation. vero who, huh . , and calu- cells are also permissible cell types for sars-cov- infection and replication, however a cells do not appear to be suitable for high levels of sars-cov- replication. our results also suggest that huh cells appear to be permissible for sars-cov- infection and replication, but do not appear to be suitable for egress into the cell culture supernatant. due to ongoing sars-cov- vaccine development efforts, there is an urgent need to develop and evaluate the susceptibility of small animal models to sars-cov- infection and covid- . recent studies have suggested that rodents may be used for these purposes, as well as to study the adaptive immune response to sars-cov- infection [ ] [ ] [ ] [ ] . to this end, we next sought to determine permissivity of rodent cell lines to sars-cov- infection, namely t and shhc cell lines. figure c shows the sars-cov- genome copy number for whole cells for murine and hamster cell lines, with vero who cells included for comparison. figure d shows the sars-cov- genome copy numbers for cell culture supernatants derived from murine and hamster cell lines, with vero who supernatant included for comparison. from total cellular rna, we detected only copies/µl in t cells at hpi, the time point at which the titer peaked. similarly, with shhc cells, we detected only copies/µl at hpi, the time point at which the titer peaked. in addition to total cellular rna, we also examined supernatants from cell culture and predictably found peak titers of only copies/µl in t cells and just copies/µl in shhc cells, both at hpi. these results suggest that neither t nor shhc cell lines are suitable for supporting sars-cov- replication or egress without further experimental finally, because it is known that hace is highly expressed by intestinal epithelial cells, we sought to examine the permissivity of human gastric adenocarcinoma cell lines to sars-cov- infection [ ] . for this purpose, we used ags and mkn cell lines, and examined viral genome copies associated with both total cellular rna as well as the cell supernatant. figure e shows the sars-cov- genome copy numbers for whole cells from gastric adenocarcinoma lines, with vero who cells included for comparison. figure f shows the sars-cov- genome copy numbers for cell culture supernatants from gastric adenocarcinoma lines, with vero who supernatant included for comparison. we found that mkn cells yielded relatively high titers, with cell-associated virus peaking at . × copies/µl at hpi. virus in the supernatant peaked at . × copies/µl at hpi. ags cells yielded lower titers, with cell-associated virus peaking at . × copies/µl at hpi and virus in the supernatant peaking at . × copies/µl hpi. interestingly, however, the titer in ags cells appeared more variable compared to other time points, increasing at and hpi and dropping at and hpi. these results suggest that these gastric adenocarcinoma cell lines can support infection, replication and egress of sars-cov- as well as, or in some cases better than, vero cell lines. given that our studies conducted to quantify sars-cov- viral genome copies in susceptible cell lines yielded results that highlighted highly permissive cell lines, like vero who, while also distinguishing less permissive cell lines, like a , we sought to analyze spike and hace protein co-expression to determine if higher hace expression correlated with higher susceptibility to sars-cov- infection. one facet of our understanding of the current sars-cov- outbreak that is rapidly evolving is sars-cov- seroprevalence in the general population. at the same time, forming a better understanding of and ability to assess the kinetics of the neutralizing antibody response to sars-cov- could be essential in further vaccine and anti-viral development efforts. to this end, we adapted the sars-cov- ffa for the quantification of neutralizing antibody (nab) titers in the form of an frnt. this was accomplished by incubating serially diluted convalescent serum from sars-cov- infected individuals with a known quantity of infectious sars-cov- (~ ffu) and measuring foci formation. infection was normalized to a pbs control to reflect the percent neutralization of sera. first, we sought to determine whether the frnt could be used to detect a range of nab concentrations in human samples. figure a shows the neutralization curves for human sera samples, showing a decrease in virus neutralization as the serum is diluted out. these samples were collected from human subjects at the university of puerto rico following a positive qpcr test for sars-cov- . all subjects were in the convalescence period at the time of sample collection. these assays were performed using deidentified residual sera samples. using the frnt approach, we quantified the neutralizing antibody titer in the form of the reciprocal serum dilution required to neutralize % of virus, or the frnt value. this assay can also be used to determine the frnt value (i.e. required for % neutralization). the frnt and frnt values are reported in figure b . the reciprocal serum dilutions required for % neutralization for the human samples (hs_a, hs_c and hs_d) are . , . , and . , respectively. the reciprocal serum dilutions required for % neutralization for hs_a, hs_c and hs_d are . , . , and . , respectively. for hs_b, the reciprocal serum dilution required to neutralize both % and % of the virus was below the lower limit of quantitation (lloq) for the assay. in order to increase confidence that these nabs were the result of recent sars-cov- infection rather than cross-reactivity with the four circulating human common cold coronaviruses, we performed an elisa to examine binding of these sera samples to sars-cov- receptor- binding domain (rbd). figure c shows the absorbance at nm (a ) values indicating that sera from these subjects contain antibodies that can bind specifically to the receptor binding domain (rbd) of sars-cov- . having confirmed that our assay can detect nab to sars-cov- in human sera, we next sought to demonstrate that this assay is applicable for numerous sera sources including non-human primates and mice. to this end, we performed an frnt with non-human primate (nhp) sera, which consisted of pooled sera samples from a group of rhesus macaques in the convalescent phase following sars-cov- infection by multiple routes (bei: nr- ). figure e shows the neutralization curve for this pool of nhp sera. low but detectable nab titers were present in this sample with an frnt value of . and frnt of . , as depicted in figure f . having shown that our assay can be utilized to quantify nabs in nhp samples, we next sought to demonstrate that this assay could also be used for quantifying nabs in small animal models such as mice. this also afforded us the opportunity to examine the nab response both at having demonstrated that our frnt assay can be used to quantify nab titers for human samples, non-human primates, and mice both in acute infection and memory responses, we next sought to determine whether this assay could be used to quantify nab resulting from a subunit or dna vaccine. to this end, we immunized c bl/ mice intramuscularly (i.m) with µg of dna encoding the sars-cov- spike (ms_c). a subset of these mice was boosted days later (ms_b, ms_d) with µg of dna intramuscularly and sera collected days following the boost. figure g shows the neutralization curves for these mice immunized with dna encoding the immunization. from these data we were able to define the frnt values for ms_b, ms_c, and ms_d, which are shown in figure h , and are . , . , . , respectively. however, the frnt values were below the lloq for the assay, as well as the frnt value for ms_a. these results suggest that the frnt can be used to detect nabs in sera resulting from immunizations, in addition to nabs in human, nhp, and mouse sera resulting from sars-cov- infections. to address the need for high-throughput, rapid quantification of infectious sars-cov- , our group has developed a focus forming assay (ffa) for sars-cov- using vero who cells. the strength of the ffa is the rapid visualization of individual foci forming from a single infectious unit or focus forming unit (ffu). the ffa for sars-cov- can be developed in as little as hours, shorter relative to traditional plaque assays for human coronaviruses which can take - days [ , , ] . the focus forming assay is also amenable to a -well plate format, allowing for assays to be scaled up or automated to handle large volumes of samples quickly relative to assays requiring plates with wells or fewer. automating the quantification of foci using equipment such as a ctl machine can also streamline the process of screening large numbers of samples. one potential disadvantage of the focus forming assay is the requirement of a sars-cov- specific antibody as the primary antibody for foci immunostaining. however, for our assays we have found that polyclonal guinea pig serum provides reproducible staining with minimal background when used at the appropriate concentrations, and numerous human monoclonal antibodies are now commercially available and suitable for this purpose [ ] [ ] [ ] . in regard to the focus forming assay development, we initially hypothesized that the absence of in vero e cells would make them more susceptible to sars-cov- infection and therefore a more sensitive choice of cell line for the focus forming assay. surprisingly, we found that vero who cells were more suited to foci formation. it is worth noting that other labs have shown that a higher titer and larger, clearer plaques result when vero e cells are used in place of vero who cells when performing plaque-assay based titrations with sars-cov- [ ]. this may reflect differences between the wuhan clinical isolate used in this study as opposed to the usa- wa / isolate or this may be an artifact of the focus forming assay. because we find that by qpcr, genome copy number is typically highest in vero e cells, we hypothesize that more defective or non-infectious virus results from replication in vero e cells. additionally, high levels of genome replication in vero e may not correlate with ability to spread laterally in cell culture and form foci. the discrepancy in sars-cov- replication in these two cell lines warrants further study. our understanding of the impact of cell type on sars-cov- entry, replication, assembly, and egress is in its infancy. these gaps in our knowledge were recently made evident by the use of chloroquine and hydroxychloroquine-widely used anti-malarial drugs that create suboptimal to advance our understanding of the sars-cov- life cycle in susceptible cell types, we generated multi-step growth curves for a variety of human, simian, and rodent cell types. in most cases, viral replication peaked at hpi in susceptible cell lines and this cell-associated virus was maintained for the duration of the experiment. in many cases, however, the presence of virus in the supernatant did not peak until - hpi. in the context of the viral replication cycle, our data suggests that genome replication in vitro peaks after just hours, however assembly and egress from infected cells may take as long as - hpi. while there are conflicting reports concerning the suitability of huh cells for sars-cov- studies [ , ] we observed a striking discrepancy was between cell-associated virus within the total rna and the virus detected within the cell supernatant. as much as . × copies/µl of cell-associated virus was detectable in rna isolated from huh cells, but virtually no detectable virus was found in the cell supernatant. this suggested to us that viral entry-and hence the production of cell-associated virus within the total rna fraction-was independent of successful viral egress. this trend did not hold for rig-i-deficient huh . cells [ ] , suggesting that viral egress is interferon (ifn) sensitive. this observation is in accordance with previous studies describing sars-cov- sensitivity to type i ifns [ , ] . further studies are warranted to determine what factors are necessary and sufficient for viral egress and could therefore serve as potential therapeutic targets. cov- infection, pathogenesis, and possibly transmission [ , [ ] [ ] [ ] , which may reflect differing susceptibility of hamster cell types based on anatomical location of the isolated cells. we did not observe a strong correlation between ace and viral spike protein levels, nor did we see a strong relationship between viral genome copy and ace mrna level. our results suggest that host cell susceptibility to sars-cov- infection is more complicated than ace expression alone, thus warranting further investigation. we have showed by elisa that convalescent sera sourced from human, non-human primate, and mice infected with sars-cov- can bind to the rbd of sars-cov- . while the s subunit of the sars-cov- spike protein is highly conserved among betacoronaviruses, previous studies have showed that the rbd within the spike protein of sars-cov- is unique [ , ] . in serological studies of sars-cov- , the presence of antibodies binding sars-cov- rbd is considered the most sensitive and specific indicator of previous sars-cov- exposure. these results increase our confidence that within these polyclonal sera samples are neutralizing antibodies that are specific to sars-cov- , rather than cross-reactivity due prior coronavirus exposure, as has been called into question by some [ , , ] . as other labs have noted [ ] we observed that binding to sars-cov- rbd appears to correlate with neutralization capacity, as human samples with a high auc for rbd binding by elisa also had lower ec values, indicating that low concentrations of sera from these patients were sufficient to neutralize % of a standardized amount of virus. we have showed that each of these samples can effectively neutralize sars-cov- in vitro and that neutralization can be with µl per well of diluted samples. this plate containing sample dilution on the cell monolayer was placed in an incubator with °c, % co for hour. a solution of % methylcellulose (sigma-m - g) in × pbs was made in advance of the assay and stored at °c until ready to use. on the day of the assay and during the one-hour infection period, % methylcellulose was diluted : in % dmem and placed on a rocker to mix. the % methylcellulose-media mixture (hereby referred to as overlay media) was stored at room temperature until ready to use. after the one-hour infection period, the well plate containing sample dilution and cell monolayer was removed from incubator. overlay was added to the plate by adding µl of overlay media to each well. this step reduces the uncontrolled spread of virus throughout the monolayer on the well, making it difficult to distinguish individual foci. after the addition of overlay media, the plate was returned to an incubator with °c, % co for hours. the plate was removed from the incubator and the media containing the overlay and sample was aspirated off. one wash with µl of × pbs per well was performed, taking care not to disrupt the cell monolayer. µl per well of % pfa in pbs was added for the fixing step. with the % pfa still in the plate, the plate was submerged in a bath of % formalin buffered phosphate (fisher: sf - ) in × pbs for minutes at room temperature. after minutes, the plate was removed from the formaldehyde bath and the % pfa removed from the monolayer. one wash with µl of × pbs (tissue culture grade) per well was performed. the plate was submerged in a bath of × pbs to rinse and removed from bsl- containment. foci were visualized by an immunostaining protocol. the -well plate was first washed twice with µl per well of ffa wash buffer ( × pbs, . % triton x- ). the primary antibody consisted of polyclonal anti- sars-cov- guinea pig sera (bei: nr- ) and was diluted : , with ffa staining buffer ( × pbs, mg/ml saponin (sigma: )).then, µl per well of primary antibody was allowed to incubate for hours at room temperature or °c overnight. the -well plate was then washed three times with µl per well of ffa wash buffer. the secondary antibody consisted of goat anti-mouse conjugated horseradish peroxidase (sigma: a- ) diluted : , in ffa staining buffer. similarly, µl per well of secondary antibody was allowed to incubate for hours at room temperature or °c overnight. the plate was washed three times with ul per well of ffa wash buffer. finally, µl per well kpl trueblue hrp substrate was added to each well and allowed to develop in the dark for - minutes, or until blue foci are visible. the reaction was then quenched by two washes with millipore water and imaged immediately thereafter with a ctl machine to quantify foci. briefly, sera samples were diluted : in % dmem and added to the topmost row of a round bottom -well plate. sera samples were then serially diluted : down the remainder of the plate in % dmem. an equal volume of sars-cov- diluted to ~ ffu/ml (~ ffu/ µl) was then added to the serially diluted sample, mixed thoroughly, and allowed to incubate at °c for hour. then µl of sars-cov- +sera mixture was transferred to a vero who cell monolayer (as described in the focus forming assay). from this point, the assay was as described in the focus forming assay section. binding of human polyclonal sera to recombinant sars-cov proteins was determined by elisa. a ug/ml mixture of µl per well containing recombinant protein in carbonate buffer ( . m na co . m nahco ph . ) was used to coat maxisorp (thermofisher) -well plates overnight at °c. plates were blocked with blocking buffer (pbs + %bsa + . % tween) for hours at room temperature the following day and washed four times with wash buffer. polyclonal sera was serially diluted in blocking buffer prior to plating. sera was allowed to incubate for hour at room temperature and washed four times with wash buffer. following the one hour incubation, goat-anti-human igg hrp (sigma) conjugated secondary ( : ) was added and allowed to incubate for hour at room temperature. the plate was washed again four times with wash buffer and the elisa was visualized with µl per well of tmb enhanced substrate (neogen diagnostics) and allowed to develop in the dark for minutes. a solution of n hcl was used to quench the reaction and the plate was read for an optical density of nanometers on a biotek epoch plate reader. the total peak area under the curve (auc) was calculated using graphpad prism . isolation of rna from cell culture and culture supernatants rna was isolated from cell culture and supernatant using an invitrogen purelink rna mini kit according to the manufacturer's instructions. rt-qpcr hace expression was measured by qrt-pcr using taqman primer and probe sets from idt (assay id hs.pt. . ). sars-cov- viral burden was measured by qrt-pcr using taqman primer and probe sets from idt with the following sequences: were allowed to infect cell monolayer for hour at °c, % co before overlay with % methylcellulose in % dmem. following a hour incubation at °c, % co , cells were fixed in a solution of % paraformaldehyde diluted in × pbs. foci were visualized via immunostaining with polyclonal guinea pig anti-sars-cov- sera and goat anti-guinea pig conjugated hrp. kpl neutralization capacity was measured by mixing serially diluted sera with a standardized amount of virus (~ ffu) and allowed to incubate for hour at °c, % co before allowed to infect a cell monolayer as described in figure non-human primate, and mouse sera samples previously described. the incubation period of coronavirus disease publicly reported confirmed cases: estimation and application. annals of internal medicine early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia identification of a novel coronavirus causing severe pneumonia in human: a descriptive study a new coronavirus associated with human respiratory 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mask partition reduces the risk of non-contact transmission in a golden syrian hamster model for coronavirus disease (covid- ) a serological assay to detect sars-cov- seroconversion in humans cross-reactivity towards sars-cov- : the potential role of low-pathogenic human coronaviruses. the lancet microbe human neutralizing antibodies elicited by sars-cov- infection key: cord- -oquk t authors: park, jung-eun; cruz, deu john m.; shin, hyun-jin title: clathrin- and serine proteases-dependent uptake of porcine epidemic diarrhea virus into vero cells date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: oquk t porcine epidemic diarrhea virus (pedv), a member of the genus alphacoronavirus, is a causative agent of porcine enteric disease characterized by acute watery diarrhea and dehydration in sucking piglet. similar to other coronaviruses, pedv spike protein mediates its cell entry by binding to cellular receptors and inducing membrane fusion between viral envelopes and cellular membranes. however, the entry mechanism of pedv is not studied. here, we determined the entry mechanism of pedv into vero cells. our data confirmed that pedv entry followed clathrin-mediated endocytosis independence of caveolae-coated pit assembly. the internalized pedv was co-localized with the clathrin-mediated endocytic marker, but not with the caveolae-mediated endocytic marker. in addition, cells treated with lysosomotropic agents and serine protease inhibitors were resistant to pedv. our data revealed that pedv entry followed clathrin-mediated endocytosis and was dependent on a low ph and serine proteolysis for successful entry into cells. infection of enveloped viruses is initiated by binding of surface proteins with specific receptor(s) on the surface of the cell membrane, which leads to internalization of the virus into cells. the second step of infection following virus attachment is the uncoating of the viral genome into the cytoplasm after the viral envelope has fused with the host membrane. there are two major routes for enveloped viruses to enter host cells; the non-endosomal and the endosomal pathways (pelkmans and helenius, ; smith and helenius, ) . both pathways require the release of the viral genome by fusion of the viral envelope with the respective target membrane of the host cells such as the plasma or endosomal membrane, respectively (matlin et al., ) . in the non-endosomal pathway, the viral envelope directly fuses with the plasma membrane. membrane fusion is mediated by a conformational change of the viral glycoprotein, which is induced by its interaction with the corresponding receptor on the host cell surface and/or proteolytic processing (blumenthal et al., ) . the endocytic pathway can further differentiate into two well-characterized pathways; those acting via the clathrin-coated pit and the caveolae-mediated lipid raft (brodsky et al., ; pelkmans and helenius, ) . after internalization, viruses require a low-ph environment in the endosome to trigger conformational changes in the viral glycoproteins. the acidic ph environment is also important for proteolytic activation of viral glycoproteins by endosomal proteases (qiu et al., ; simmons et al., ) . the porcine epidemic diarrhea virus (pedv) is classified as alphacoronavirus together with transmissible gastroenteritis virus (tgev), feline infectious peritonitis virus (fipv), and human coronavirus e (hcov- e). pedv causes an acute watery diarrhea in suckling piglets, which results in approximately % mortality among suckling piglets and reduces the weight among fattening pigs (debouck and pensaert, ) . porcine epidemic diarrhea (ped) is first recognized in pigs in the united kingdoms in (wood, ) . although no evidence of ped is currently reported from canada, similar coronavirus-like particles were reported from herds in quebec in (turgeon et al., ) . since then, outbreaks of ped have been documented in many european and asian countries such as czech republic, hungary, korea, the philippines, china, italy, thailand, germany, spain, and japan (song and park, ) . recently, pedv is spreading rapidly in swine farms in the united states, resulting in high mortality in piglets in more than states (mole, as typical for the alphacoronavirus, the pedv spike (s) protein encounters virus entry into host cells by interacting with its receptor, porcine aminopeptidase n (apn), in porcine enterocytes and by mediating membrane fusion with host cell membranes (li et al., ; oh et al., ) . upon receptor binding, several coronaviruses in alphacoronavirus enter cells via endocytosis. for example, extensive studies on hcov- e have shown that upon binding with the human apn receptor, it is taken up in lipid rafts and enters via caveolae-dependent endocytosis (nomura et al., ) . inside the endosome, cellular proteases that are active in a low-ph environment facilitate membrane fusion (kawase et al., ) . similarly, tgev binds to porcine apn (weingartl and derbyshire, ) , and has been shown to enter mdck cells over-expressing porcine apn via endocytosis and acidification of the intracellular compartment facilitated membrane fusion (hansen et al., ) . fipv also requires acidification of endosomes for successful entry (takano et al., ) . inhibition of fipv infection with nystatin, a pharmacological reagent that causes caveolae to flatten and disrupt the coat structure, and dynamin inhibitor suggests that fipv entry might actually involve some types of caveolae-dependent endocytosis (van hamme et al., ) . although several studies have examined the mechanism of entry of other coronaviruses, the mechanism of pedv entry is still unknown. in this study, we studied the entry mechanism of pedv by measuring virus infectivity in the presence of chemical inhibitors and co-localization of pedv with endocytic pathway markers. we found that pedv infection was diminished by treatment with chloropromazine (cpz) and lysosomotropic agents. in addition, we also investigated that pedv required serine-like proteases for their entry through endocytosis and for cell-cell fusion. taken together, our findings reveal that pedv enters vero cells via clathrin-mediated endocytosis and requires serine proteolysis during infection. vero cells were maintained in eagle's minimum essential medium (mem, gibco) containing with % heat-inactivated fetal bovine serum (fbs, gibco), u/ml penicillin, g/ml streptomycin and g/ml amphotericin b. kpedv- , a vero cell-adapted korean strain, was propagated in vero cells as described previously (hofmann and wyler, ) . briefly, vero cells were inoculated with the kpedv- at a multiplicity of infection (moi) of and cultured in serum-free mem for h at • c with % co . the progeny viruses were titrated using the focus formation assay following a method described previously (cruz and shin, ) . kpedv- infection in vero cells under trypsin and non-trypsin conditions was compared for h. vero cells in -well tissue culture (tc) plate (spl labware) were inoculated with kpedv- and cultured in either serum-free mem or mem supplemented with trypsin ( g/ml). infection was stopped by addition of % paraformaldehyde (pfa) at the indicated times for immunocytochemistry. vero cells were treated with various concentrations of either cpz for min or . m sucrose for min to inhibit the formation of clathrin-coated pits. to block the caveolae-dependent pathway, cells were incubated with various concentrations of nystatin for min. control cells were incubated with or without dimethyl sulfoxide (dmso). cells were inoculated with kpedv- at a moi of for h, and then overlaid with . % methyl cellulose in mem containing trypsin for h. at hpi, pedv-infected cells were detected by immunocytochemistry. to prepare ultra-purified trypsin-free viruses, vero cells were inoculated with the kpedv- at a moi of and cultured in serumfree mem for h. supernatant was clarified by centrifugation at , × g for min at • c, followed by ultra-centrifugation using a % sucrose cushion at , × g for . h. following resuspension in buffer a ( m tris, ph , m nacl, . m cacl ), protein concentration of purified virus stock was determined by the bradford assay. fluorochrome conjugation of kpedv- with alexa fluor (af ) carboxylic acid-succinimidyl ester (molecular probes) was performed according to manufacturer's instructions. briefly, . mg of ultrapurified kpedv- was dialyzed in labeling buffer ( . m nahco , ph . ) at • c overnight. virus was then incubated for h on a platform rocker at room temperature with g of af succinimidyl ester in l of dmso. the af -labeled kpedv- was extensively dialyzed in buffer a. vero cells were prepared on cover glasses a day before assay. for af -kpedv- co-localization with endocytic markers, the cells were incubated with af -kpedv- combination with g/ml of alexa fluor -conjugated transferrin (af -tf) or . g/ml of alexa fluor -cholera toxin subunit b (af -ct-b) for min on ice to synchronize entry, and then shifted to • c. unbound viruses were removed, and the cells were fixed in % pfa at indicated times and analyzed at magnification of × on the laser scanning confocal microscope. vero cells were treated with either mm nh cl or g/ml baf-a to neutralize the intracellular ph. the cells were then inoculated with kpedv- at a moi of for h in the presence of lysosomotropic agents. the virus inoculums were removed by washing with pbs. cells were then overlaid with . % methyl cellulose in mem containing trypsin for h. at hpi, pedv-infected cells were detected by immunocytochemistry. the effect of low ph on the fusion activity of the s protein was investigated by subjecting pedv-infected vero cells to a low ph range. vero cells were inoculated with kpedv- and cultured in trypsin-free mem for h. afterwards, the cell monolayer was washed thrice with pbs and replenished with serum-free mem adjusted to ph , , , , . mem containing trypsin ( g/ml) at ph was used as positive control. the cultures were further incubated at • c for h, and then fixed with % pfa. pedv-infected cells were detected by immunocytochemistry. cells were pretreated with various protease inhibitors such as e- ( m), aebsf-hcl ( m), pepstatin a ( g/ml), and phosphoramidon ( m) for h. for examination the synergistic antiviral activity of aebsf-hcl and lysosomotropic agent, cells were treated with aebsf-hcl and/or nh cl for h. treated cells were then infected with kpedv- at a moi of for h in the presence of inhibitors. after h adsorption, virus inoculums were removed by washing with pbs. cells were then overlaid with . % methyl cellulose in mem containing trypsin for h. at hpi, pedv-infected cells were detected by immunocytochemistry. vero cells were pretreated with either mm nh cl or g/ml cpz and then inoculated with kpedv- at a moi of for h. after adsorption, virus inoculums were removed by washing with pbs. cells were then overlaid with . % methyl cellulose in mem containing trypsin for h. at hpi, pedv-infected cells were detected by immunocytochemistry. to detect expression of viral proteins, kpedv- infected vero cells were fixed with % pfa for min and permeabilized with % np . following three washes with pbs, cells were incubated with : dilution of mouse anti-pedv polyclonal antibody for h. cells were washed three times with pbs and then incubated with : dilution of goat anti-mouse igg conjugated with horseradish peroxidase. kpedv- infected vero cells were stained using a , -diaminobenzidine tetrahydrochloride solution containing nicl and h o (vector laboratories). clusters of immunostained cells were observed under the inverted microscope (zeiss) and were presented as the ratio between mock-treated and dmso treated cells. vero cells prepared in -well tc plates were treated with various chemicals to inhibit each endocytic pathway as described above. cells were inoculated with kpedv- at a moi of in the presence of drugs. at hpi, unbound viruses were washed out and then cells were incubated in serum-free mem. infected cells were harvested and lysed in pro-prep protein extraction solution (intron) at hpi. the extracted proteins were diluted in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) sample buffer and electrophoresed in % sds-page system. separated proteins were electrically transferred onto a polyvinyl difluoride membrane and pedv n protein was traced using anti-pedv polyclonal antibodies. bands were visualized using supersignal west dura with las- plus. it has been reported that pedv requires an extracellular trypsin for its successful infection in vitro (kusanagi et al., ; lai and cavanagh, ; park et al., ) . early data on the infection of pedv were first provided by hofmann and wyler when they demonstrated the formation of multinucleated cells in pedvinfected vero cells by supplementing the culture media with trypsin (hofmann and wyler, ) . the trypsin-induced syncytium formation is corroborated by li et al. when papn-expressing mdck cells were infected with pedv in the presence of trypsin (li et al., ) . previous findings suggest that proteolytic processing of the s protein is required to facilitate viral membrane fusion with cellular membranes. and, it also raises the possibility that pedv entry by direct fusion with the plasma membrane could take place in the presence of trypsin. to confirm the role of exogenous proteases in pedv infection, pedv infected cells were treated with trypsin and evaluated viral infectivity by immunocytochemistry. as shown in fig. , we found that pedv could infect vero cells even without trypsin treatment. initial infection was confirmed as early as hpi on both trypsin and non-trypsin conditions. the number of infected cells in both conditions was similar but the spreading into adjacent cells was faster in the presence of trypsin. at hpi, more than % of the cell monolayer had formed large multi-nucleated cells. in sharp contrast, pedv growth without exogenous trypsin did not involve syncytial spread. at hpi, more than % of the cells showed signs of infection, but still no cytopathic effect (cpe) was found. these results confirm that trypsin catalyzes pedv s protein-mediated cell-cell fusion as demonstrated previously (hofmann and wyler, ; park et al., ) . based on these observations, we concluded that an exogenous protease, like trypsin, was necessary to induce cell-cell fusion in pedv-infected vero cells but not essentially required for virus-cell entry. so, we hypothesized that pedv entry into vero cells under the trypsin-free condition most likely occurred inside endosomal compartments where cellular proteases might operate similar to trypsin, facilitating s-mediated fusion of pedv with the endosomal membrane. the enveloped virus entry through endocytosis can differentiate into two well-characterized pathways; those acting via the clathrin-coated pit and the caveolae-mediated lipid raft (brodsky et al., ; pelkmans and helenius, ) . to explore whether the endocytic pathway supports pedv entry into vero cells and which endocytic pathway alters for pedv infection, we inhibited pathway by using substances interfering either clathrin-mediated endocytosis or caveolae-mediated endocytosis. for the inhibition of clathrin-mediated endocytosis, we used either (i) cpz, which is known to abolish the formation of clathrin-coated vesicles by interfering with the interaction of the adapter protein ap- with the clathrin-coated pit lattice and thus inhibiting clathrindependent endocytosis or (ii) hypertonic . % sucrose, which inhibit clathrin-mediated endocytosis by inducing dispersion of clathrin lattices on the plasma membrane. for the inhibition of caveolae-mediated endocytosis, we used nystatin, a polyene antifungal agent that interacts with cholesterol and inhibits the lipid raft/caveolin pathway. concentrations of substances were chosen according to previous studies showing the inhibition of other enveloped viruses entering the cells via thee these endocytic pathways. to access the inhibitory effect of cpz on pedv infection, vero cells were treated with cpz and infected with kpedv- . to measure the inhibitory effect of virus entry, cells were overlaid with . % methyl cellulose in mem containing trypsin for h. media containing methyl cellulose and trypsin blocks second-cycle infection, but allows the formation of syncytium to visualize infected cells. infectivity was determined by measuring infected cells by immunocytochemistry staining at hpi and normalized with "untreated cells" control. as shown in fig. a , pedv infection remarkably diminished (> %) by cpz treatment, and the inhibition rate was positively related with concentration of cpz. to confirm decreased replication of pedv, we determined the expression of pedv nucleocapsid (n) proteins by western blotting. the pedv n proteins were far less expressed in cpz-treated cells compared to untreated cells (fig. b) , whereas ␤-actin expression was same. consistent with previous results, pedv infection with hypertonic sucrose treated was also significantly inhibited (fig. c ). our result strongly suggested that pedv uses clathrin-mediated endocytosis pathway for their entry into vero cells. similar experiments were performed with nystatin treatment to determine whether pedv also uses caveolae-mediated endocytosis. unlike cpz treatment, pedv infection was only slightly decreased in highest concentrations (fig. a) . likewise, the levels of pedv n protein synthesized virtually identical in both presence and absence of nystatin (fig. b) . to verify and confirm our results, several other markers specific to the clathrin-mediated pathway or caveolae-mediated endocytosis were used to provide direct evidence that pedv uses this pathway for entry. we traced and visualized pedv location in vero tf is transported into the cells in a vesicle by receptor-mediated clathrin-dependent endocytosis pathway. ct-b specifically binds to glycolipid monoganglioside at the plasma membrane and is internalized and delivered to the golgi complex through caveolaemediated endocytosis pathway (nichols, ) . vero cells were incubated with fluorescence-labeled pedv along with each marker, and then evaluated their subcellular localizations by confocal microscopy. at h later, both endocytic markers, ct-b and tf, were located in the cytoplasm. co-localization was observed only between pedv and tf (fig. a ), but not with ct-b (fig. b) . to further confirm these findings, vero cells were treated with cpz and nystatin prior to pedv inoculation. as shown in fig. c , pedv and tf were co-localized in the cytoplasm in both nystatin and mock-treated, but not in cpz-treated vero cells. both pedv and tf were found only on cell surface indicating that clathrin-mediated and tf (green, a), but not between kpedv- and ct-b (green, b), was observed in the merged images. (c) colocalization of (yellow) kpedv- and tf was observed inside cells in the mock-and nystatin-treated vero cells. by contrast, cpz treatment inhibited internalization of both kpedv- and tf. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) endocytosis was successfully (or completely) inhibited by cpz treatment. taken together, we confirmed and concluded that the clathrin-mediated endocytosis pathway was important for pedv entry. we next explored whether pedv infection requires the acidic environment in endosomal compartments. for the inhibition of endosomal acidification, vero cells were treated with either nh cl, a relatively weak base accumulating inside endosomal vesicles, or baf-a , specific inhibitors of the vacuolar h+-atpase in animal and other eukaryotic cells. neutralization of ph in acidic organelles was confirmed by a fluorescent ph indicator probe, lysosensor (data not shown). we evaluated the inhibitory activities of different lysosomotropic reagents by measuring infected cells. both lysosomotropic reagents showed strong inhibitory effects on production of progeny pedv, especially with % reduction at hpi output titer by nh cl (fig. a) . pedv replication was about % inhibited at concentrations as low as nm of baf-a . these results indicated that pedv entry was very sensitive to low ph and acidic condition in endosome and/or late endosome might be critical for its entry. for further observation whether acidification solely induce pedv s-mediated fusion, we evaluated pedv s-mediated cell-cell fusion in acidic conditions. as shown in fig. b , low ph did not induce cell-cell fusion. syncytium formation was not observed in any cells under the condition between ph and ph (panels a-d). similarly, no syncytium formation was found under neutral ph condition (panel e). this is in stark contrast to the cell-cell fusion observed after the addition of trypsin in the culture media at neutral ph (panel f). collectively, we concluded that the acidic condition is important but still not sufficient for pedv s-mediated membrane fusion. and also, the fusogenic property of s protein could be activated by proteolytic processing. to evaluate the role and effect of proteases other than trypsin in pedv entry, we used protease inhibitors. first of all, we checked cytotoxicity of all those inhibitors to exclude false results with recommended concentrations, and confirmed no cell damaged by them (data not shown). as shown in fig. a , we found aebsf-hcl induced the strongest inhibitory activity with more than % inhibition. in contrast, e- , pepstatin a, and phosphoramidon showed relatively lower inhibition with about - % inhibition. these results suggested that serine proteases were importantly involved in pedv entry into vero cells. for the cases of severe acute respiratory syndrome-associated coronavirus (sars-cov) and mhv- , it has been reported that phdependent endosomal cellular factors were required for proteolytic activation of s proteins, rather than the virus requiring an acidic trigger itself (qiu et al., ; simmons et al., ) . to evaluate this for pedv s case, cells were treated with aebsf-hcl in combination with nh cl prior to infection. as shown in previous results, pedv infection was significantly inhibited either by aebsf-hcl or nh cl treatment (fig. b) . the inhibitory effect on combination treatments by both was similar to that of aebsf-hcl single treatment. s protein activation by exogenous proteases renders coronavirus s mediated virus-cell fusion independent of cathepsin activity. finally, we determine whether trypsin treatment bypass entry inhibition by endocytosis inhibitors. vero cells were pretreated with either cpz ( g/ml for min) or nh cl ( mm for h) prior to virus infection and then infected with pedv in the absence or presence of trypsin. pedv infectivity was only slightly facilitated by trypsin treatment and trypsin treatment does not overcome the inhibitory effect of nh cl and cpz. the small increases of pedv infectivity might be obtained by rapid spreading of virus infection via cell-cell fusion. these results suggested that trypsin did not support proteases-mediated virus-cell entry. the entry mechanism of pedv, a coronavirus, is largely unknown. here, to examine the entry pathway of pedv into vero cells, we used essentially two independent and complementary approaches: (i) the focus formation assay to assess the level of infection by viruses in cells treated with various inhibitors, and (ii) fluorescence microscopy to monitor the entry of viruses into cells along with well-established markers. both approaches provided similar conclusions on the mechanism of pedv cell entry. the infection inhibition assay using various substrates that interfere with endocytosis or lysosomotropic agents revealed that pedv enters vero cells via clathrin-mediated endocytic uptake and delivery of virus to an acidic intracellular compartment. more interestingly, we found that pedv requires serine proteolytic processes in early stages of infection. the serine proteolysis activates pedv entry in independent manner with acidic ph environment, but did not bypass the infection reduction by lysosomotropic agents. in the cell entry of many coronaviruses, the proteolytic activation of s proteins triggers viral membrane fusion and essentially required for virus entry (huang et al., ; qiu et al., ; simmons et al., ) . cathepsins, which is a family of cysteine proteinases commonly found in acidified endosomes, have been associated with the proteolytic processing of s glycoproteins in sars-cov, mhv- , and hcov- e and mediated viral membrane fusion with endosomal membranes within endosomes (kawase et al., ; qiu et al., ; regan et al., ; simmons et al., ; turk et al., ) . similarly, various exogenous and cellular proteases such as trypsin, transmembrane proteases serine enhance sars-cov entry by inducing virus-cell fusion at cell surface (glowacka et al., ; matsuyama et al., matsuyama et al., , shulla et al., ) . the block to infection mediated by lysosomotropic agents could be bypassed by treating with exogenous or cellular proteases (matsuyama et al., ; simmons et al., ) . pedv infection in vitro is also largely dependent on trypsin supplement (hofmann and wyler, ; park et al., ) . it raises possibility that pedv s could fuse with plasma membrane by activation with proteases to deliver their genomes into cells. however, our study demonstrated that pedv entry occurs without exogenous proteases. although pedv infection was likely facilitated by trypsin treatment as demonstrated earlier, pedv also propagated even without trypsin (fig. ) . based on our results, we could confirm that exogenous protease, especially trypsin, might be critical factor for cell-cell fusion but not for viral envelope-cell membrane fusion. and also, we could conclude that trypsin is not essentially required for virus entry into vero cells. our results encouraged us to hypothesize that pedv penetration must have been facilitated by fusion of its envelope with the host membrane in a fusion-permissive environment, which most likely occurs inside endosomal compartments. pedv might take endosomal entry pathway rather than direct fusion. the experiment using various inhibitors supported our hypothesis that pedv alters endosomal pathway for their entry. pedv infection was greatly diminished by pre-treatment with cpz and hypotonic sucrose (fig. ) . it suggests that pedv enters vero cells via clathrin-mediated pathway. furthermore, co-localization between endocytosed tf and fluorochrome-labeled pedv may support conclusion that clathrin mediated endocytic uptake is major pathway for entry (fig. ) . our data collectively propose that pedv enters vero cells via clathrin-mediated endocytosis similar to other coronaviruses. vero cells were treated with lysosomotropic agents, either nh cl or baf-a , and then infected with kpedv- . pedv entry was scored by immunocytochemistry at hpi. the relative infectivity was showed as percentages of infected cells to untreated cells. the error bars represent standard deviations of the mean values. (b) low ph does not convert the pedv s protein to its fusogenic form. pedv infected vero cells were exposed to various ph conditions, and then incubated in serum-free media for h. low to neutral ph range (ph - ) did not induce cell-cell fusion of pedv-infected vero cells. in contrast, the addition of trypsin at neutral ph readily induced cell-cell fusion within h after treatment (lower right). in order to mediate membrane fusion enveloped viruses, such as influenza and dengue, the low ph of acidified endosomal compartments is sufficient for their conformational changes (plemper, ) . unlike these viruses, ph-dependent endosomal cellular factors were required for activation of low ph-dependent proteases, rather than the virus requiring an acidic trigger itself (huang et al., ; simmons et al., ) . similar to other coronaviruses, pedv infection was significantly inhibited by both nh cl and baf-a (fig. a ), but acidic ph did not induce pedv s-mediated fusion (fig. b) , suggesting that low ph did not differently mediate virus fusion and another triggering factors such as proteolytic activation might be required for successful fusion. while trypsin induce the fusion activity of pedv s proteins on the plasma membrane as demonstrated by formation of syncytia ( fig. ) , it was not clear whether similar conditions are required for fusion between its envelope and endosomal membrane. pedv entry into vero cells was specifically inhibited by serine proteases inhibitor, but not by other proteases (fig. a ). it suggests that serine or serine-like proteases in the cytoplasm are involved in facilitating the fusion between pedv envelope and host endosomal membrane. it is not clear which serine proteases are involved in pedv entry, however, we are assuming that intracellular serine proteases may cleave the s protein so that induce membrane fusion during infection. as shown in fig. b , inhibitory effect of serine proteases inhibitors was observed when the lysosomotropic reagents were treated, indicating that novel serine protease(s) is involved in pedv entry with acidic ph independent manner unlike low ph-dependent endosomal cathepsins. in addition, our finding that trypsin treatment is not bypass the infection inhibition with lysosomotropic agents and cpz as shown in fig. , clearly indicates that pedv entry requires ph-dependent step rather than the presence of ph-dependent proteolytic processing. recently, similar possibilities were proposed by others in mhv-a s mediated infection study (wicht et al., ) . another question is how trypsin sufficiently mediates cell-cell fusion at neutral ph. it was reported that the furin cleavage of s on mhv and sars-cov is required for virus-cell fusion, but not for cell-cell fusion (de haan et al., ; follis et al., ) . it seems like coronavirus s-mediated cell-cell fusion is regulated by different manner from virus-cell fusion. acidic ph and serine proteolysis are sufficient for virus-cell fusion, in contrast, serine proteolysis and/or unknown other factor(s) which may provide similar condition to low ph, are required for cell-cell fusion at plasma membranes; thus s induces membrane fusion without low ph. all our data confirmed that pedv entry followed clathrinmediated endocytosis independence of caveolae-coated pit assembly. the internalized pedv was co-localized with the clathrin-mediated endocytic marker, but not with the caveolaemediated endocytic marker. in addition, cells treated with lysosomotropic agents and serine protease inhibitors were resistant to pedv. our data revealed that pedv entry followed clathrin-mediated endocytosis and was dependent on an acidic ph and serine proteolysis for successful entry into cells. fluorescent lipid probes in the study of viral membrane fusion biological basket weaving: formation and function of clathrin-coated vesicles application of a focus formation assay for detection and titration of porcine epidemic diarrhea virus cleavage inhibition of the murine coronavirus 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spike-mediated entry differential role for low ph and cathepsin-mediated cleavage of the viral spike protein during entry of serotype ii feline coronaviruses a transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry how viruses enter animal cells porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines analysis of the mechanism of antibody-dependent enhancement of feline infectious peritonitis virus infection: aminopeptidase n is not important and a process of acidification of the endosome is necessary coronavirus-like particles associated with diarrhea in baby pigs in quebec acidic ph as a physiological regulator of human cathepsin l activity clathrin-and caveolae-independent entry of feline infectious peritonitis virus in monocytes depends on dynamin evidence for a putative second receptor for porcine transmissible gastroenteritis virus on the villous enterocytes of newborn pigs identification and characterization of a proteolytically primed form of the murine coronavirus spike proteins after fusion with the target cell an apparently new syndrome of porcine epidemic diarrhoea this research was supported by grant from the korea research foundation grant funded by the korean government (no. , (no. , - , - . we really appreciate for all the advices and technical supports from dr. fumihiro taguchi in nippon veterinary and life science university. key: cord- -xz aawem authors: mizutani, t. title: signal transduction in sars‐cov‐infected cells date: - - journal: ann n y acad sci doi: . /annals. . sha: doc_id: cord_uid: xz aawem abstract: severe acute respiratory syndrome (sars) is a newly found infectious disease that is caused by a novel human coronavirus, sars coronavirus (sars‐cov). because the mortality rate of sars patients is very high, understanding the pathological mechanisms of sars not only in vivo but in vitro is important for the prevention of sars. activation of signaling pathways caused by sars‐cov infection leads to the phosphorylation and activation of downstream molecules. two conflicting cellular programs, apoptosis to eliminate virus‐infected cells and survival to delay apoptosis by producing antiviral cytokines, occur in sars patients. recent studies regarding sars and sars‐cov have clarified that activation of mitogen‐activated protein kinases (mapks) plays important roles in upregulation of cytokine expression and apoptosis both in vitro and in vivo. both akt and p mapk are keys for determination of cell survival or death in sars‐cov‐infected cells in vitro. agents being developed to target these signaling cascades may be important for the design of anti‐sars‐cov drugs. this review highlights recent progress regarding sars‐cov biology, especially signal transduction in sars‐cov‐infected cells. severe acute respiratory syndrome (sars) is a newly found infectious disease that is caused by a novel coronavirus, sars coronavirus (sars-cov). , in late , sars spread from china to more than countries, causing severe outbreaks of atypical pneumonia. although the mechanism of sars pathogenesis in vivo may involves both the effects of viral replication in the target cells and immune responses to viral antigens, there is a lack of molecular pathological data, including data regarding the signaling pathways of sars-cov infection. as viral virulence and the mortality rate of sars patients are very high, understanding the pathological mechanisms of sars is important for the prevention of sars. generally, both proapoptotic and prosurvival signaling pathways are activated during viral replication. many pro-and antiapoptotic proteins are involved in these pathways in cells. several reports indicated that activation of physiological intracellular signaling cascades caused by sars-cov infection leads to the phosphorylation and activation of downstream molecules. for example, mitogen-activated protein kinases (mapks) are well-known signal transducers that respond to extracellular stimulation by cytokines, growth factors, viral infection, and stress, and in turn regulate cell differentiation, proliferation, survival, and apoptosis. , , recent studies regarding sars and sars-cov have clarified that activation of mapks plays important roles in upregulation of cytokine expression and apoptosis both in vitro and in vivo. this review highlights progress regarding sars-cov biology, especially signal transduction in sars-cov-infected cells (fig. ) . apoptosis, which is fundamentally different from necrosis, is an active and physiological type of cell death, which can be induced by viral infection, viral replication, and production of viral proteins. the monkey kidney cell line, vero e , is widely used in sars-cov research due to its high sensitivity to infection with the virus. several studies have shown that sars-cov infection of vero e cells induces apoptosis, detected by dna fragmentation and caspase activation. , p mapk is strongly activated by stress and inflammatory cytokines. mouse hepatitis virus (mhv), a prototype coronavirus, was able to induce activation of p mapk into virus-infected cells. p mapk activation in cd monocytes was also observed in sars patients. phosphorylation of p mapk was significantly upregulated at h postinfection (h.p.i.) in sars-cov-infected vero e cells. cytopathic effects of sars-cov-infection were partially prevented by adding the p mapkspecific inhibitor, sb , to the cells. although p mapk can promote both cell death and survival, the results of inhibitor studies indicated that the p mapk signaling pathway may be involved mainly in cell death in sars-cov-infected vero e cells. several downstream targets of p mapk were phosphorylated in virus-infected cells, and sb effectively inhibited phosphorylation of these proteins in sars-cov-infected cells. mapkactivated protein kinase (mapkapk)- , which is activated in response to stress and growth factors, , was phosphorylated in virus-infected cells. hsp- , which is a substrate of mapkapk- and is known to show antiapoptotic activity by inhibiting apoptosome formation, survival factors, camp response element-binding protein (creb), and activation of transcription factor (atf)- , was also phosphorylated in virus-infected cells. phosphorylation of hsp- , creb, and atf- may induce an antiapoptotic environment in sars-cov-infected cells. nucleocapsid (n) protein was able to induce phosphorylation of hsp- and creb in transfected cells as described below. the translation initiation factor, eukaryotic initiation factor e (eif e), is known to enhance translation rates of cap-containing mrnas. the phosphorylation of eif e was utilized to promote virus-specific protein synthesis in the case of mhv. although the levels of phosphorylated eif e were increased by sars-cov infection, the activated eif e was not advantageous for viral protein synthesis as demonstrated by the similar kinetics of viral protein accumulation in infected vero e cells in the presence and absence of sb . there may be other substrates of p mapk that are inducible on apoptosis of vero e cells caused by sars-cov infection. as each downstream target molecule of p mapk has a role in the induction of cell death or survival in response to sars-cov infection, determination of the apoptosisinducible target molecules of p mapk is important for the development of anti-sars-cov drugs. extracellular signal-regulated kinase (erk) / was phosphorylated in sars-cov-infected vero e cells, whereas erk / was downregulated in n protein-expressing cos- cells as described below. jnk c-jun n-terminal kinase (jnk) was phosphorylated in sars-cov-infected vero e cells. a recent study indicated that persistent infection was established after most of the sars-cov-infected vero e cells had died by apoptosis. sp , an inhibitor of jnk, and ly , an inhibitor of phosphatidylinositol -kinase (pi k), inhibited the establishment of persistence, whereas pd , an inhibitor of mek / , and sb , an inhibitor of p mapk, did not. thus, two signaling pathways of jnk and pi k are important for the establishment of persistence in vero e cells. in vero e cells, signal transducer and activator of transcription (stat) is constitutively phosphorylated at tyrosine (tyr)- and is slightly phosphorylated at serine (ser)- . however, sars-cov replication induced dephosphorylation of stat at tyr- . as stat is a major transcription factor activated in response to cytokines, such as interleukin- (il- ) and il- , inhibition of stat signaling by dominant negative and antisense oligonucleotides against stat resulted in decreases in cell viability and apoptosis. , thus, stat is thought to act as an antiapoptotic transcription factor. although inhibitors of mek and jnk had no effect on the phosphorylation status of stat in virus-infected cells, two inhibitors of p mapk (sb and sb ) partially inhibited dephosphorylation of stat at tyr- , suggesting that the p mapk signaling pathway is upstream of tyr- dephosphorylation of stat in vero e cells. the level of stat phosphorylation at ser- was increased in virus-infected cells. although the effect of ser- phosphorylation on the function of stat remains unresolved, it was reported that phosphorylation of ser- of stat negatively modulated its tyr phosphorylation. interestingly, tyr- dephosphorylation and ser- phosphorylation showed almost the same timing in sars-cov-infected cells. stat phosphorylated at tyr- was localized mainly in the nucleus in mockinfected cells, whereas stat disappeared from the nucleus in virus-infected cells. as stat acts as an activator of transcription in the nucleus, stat may lack its transcriptional activity in sars-cov-infected vero e cells, and thus result in a decrease in antiapoptotic activity in the cells. as other stat signal transduction pathways are involved in sars-cov infection, there have been many reports that treatment with interferons (ifns) can inhibit viral replication in vivo and in vitro. however, there have been no detailed studies of ifn signal transduction in sars-cov-infected cells. ifnalpha receptor recognizes stat and stat via jak and tyk , whereas the ifn-gamma receptor recognizes stat via jak and jak . signal-transducing adaptor molecule (stam ) was upregulated in sars-cov-infected vero e cells. as stam is known to associate with jak and via the immunoreceptor tyr-based activation motif, it may play an important role in signal transduction of cytokine receptors by sars-cov infection. as mentioned above, the pi k signaling pathway (including akt) plays important roles in establishing persistent infection by sars-cov. akt is phosphorylated at both ser- and threonine (thr)- residues via a pi k-dependent mechanism on stimulation by growth factors, insulin, and hormones. one of the most important functions of activated akt in cells is the prevention of apoptosis. akt has many downstream targets and it induces cell survival via phosphorylation of the forkhead transcription factor (fkhr) family, glycogen synthase kinase ß (gsk- ß), caspase- , and bad. , , in sars-cov-infected confluent vero e cells, ser- of akt was phosphorylated at h.p.i. and maximal phosphorylation was observed at h.p.i., whereas phosphorylation of thr- was not observed. as akt is thought to show its full activity when phosphorylated at both ser- and thr- , the total activity of akt may be low in vero e cells. therefore, the phosphorylated akt in sars-cov-infected cells cannot prevent apoptosis. interestingly, tissue inhibitor of metalloproteinase (timp ), which activates ras and leads to ras/pi k complex formation, was shown to be downregulated in virus-infected vero e cells at h.p.i. by microarray analysis. the acute ser dephosphorylation of akt at h.p.i. after tentative phosphorylation in virus-infected cells may be due to downregulation of timp . protein kinase c (pkc) is one of the major cellular mediators of biological functions. the pkc superfamily is classified as subsuperfamilies based on activation profiles: conventional pkc (cpkc ␣, ␤i, ␤ii, ␥ novel pkc (npkc ␦, ⑀, , ), atypical pkc (apkc , / ) pkc pkd and pkc. pkc , which was discovered as a unique pkc isotype, is thought to be one of the most important pkc, because pi k-dependent activation of pkc mediates bcell survival by nerve growth factor. akt has been reported to interact with pkc . pkc was phosphorylated in sars-cov-infected vero e cells, suggesting that this molecule is activated as an antiapoptotic response to sars-cov infection. human intestinal epithelial caco- cells, which are highly permissive to sars-cov, have been used for analysis of cellular gene expression by microarray analysis. the proinflammatory chemokines, interleukin (cxcl ), and interferon-␥ -inducible protein (cxcl ), were upregulated in sars-cov-infected caco- cells. these two chemokines are regulated by ap- and nf-b, which were also activated by viral infection. cxcl levels were significantly elevated in the blood of sars patients , and in macrophages in vitro. as described above and below, creb was phosphorylated in virus-infected vero e cells and n protein-expressing cos- cells. the genome of sars-cov is approximately kb in length and contains potential open-reading frames (orfs), including nine viral proteins with no homologues in other coronaviruses. the n protein, which is a amino acid predicted phospho-protein of kda with a short ser-rich stretch and a putative bipartite nuclear localization signal, is known to show little homology with molecules in other coronaviruses. the n protein has been shown to undergo self-association through the c-terminal amino acid region and was able to induce apoptosis of cos- cells in the absence of growth factors. both jnk and p mapk were upregulated in the transfected cells, whereas erk and akt were downregulated. activated p mapk by n protein induced actin reorganization into cells in the absence of growth factors. the downstream targets of p mapk, mapkapk- , and hsp- , were phosphorylated in the cells as well as in the virus-infected vero e cells. downregulation of focal adhesion kinase (fak) activity and fibronectin expression was observed due to n protein expression, supporting the hypothesis that apoptosis induced by n protein is caused by interference with the integrin signaling pathway, and that serum factors are able to inhibit apoptosis by maintaining the activity of fak through alternative pathways. caspase- and - were activated only in the absence of growth factors in n-expressing cells. however, sb , an inhibitor of p mapk, failed to inhibit caspase- activation. thus, there are two independent functional properties of n protein, p mapk-dependent actin reorganization, and caspase activation. another group showed that the levels of transcription factors, c-fos, atf , creb, and fosb, are increased by expression of n in vero and huh- cells. spike (s) protein of sars-cov also induced activation of ap- and il- promoter, possibly via activation of mapks, in a and hfl- cells. as high serum levels of il- were observed in patients in the acute stage of sars, activation of the il- promoter via ap- induced by s protein may explain these clinical observations. the binding of s protein to a viral receptor, angiotensin-converting enzyme (ace)- , may be a trigger for activation of these mapks. two unique proteins of sars-cov, a (u , x ) and a (u , x ), have been studied. the a protein is located mainly in the golgi apparatus and contains three transmembrane regions, whereas a encodes a protein of amino acids containing a probable cleaved signal sequence and a c-terminal transmembrane helix. as the c-terminal tail also contains a typical endoplasmic reticulum (er) retrieval motif, a is localized to the perinuclear region in both sars-cov-infected and -transfected cells. the overexpression of a, but not of a, induced apoptosis via a caspase-dependent pathway. in sars patients, two conflicting cellular programs occur: "apoptosis" to eliminate virus-infected cells and "survival" to delay apoptosis by producing antiviral cytokines. the control mechanisms balancing cell survival against cell death in vitro are important for understanding the pathology in vivo. as mentioned above, activation of p mapk is involved in the regulation of il- in vivo. both akt and p mapk are keys for determination of cell survival or death in sars-cov-infected cells in vitro. activation of the p mapk signaling pathway and dephosphorylation of stat via p mapk induced by sars-cov infection have partially proapoptotic roles in vero e cells. on the other hand, the levels of akt, which inactivates proapoptotic pathways, are too low to block apoptosis signaling pathways. as akt activation is necessary to establish persistently infected cells that escape from apoptotic cell death on viral infection, akt plays an important role in delaying apoptosis in sars-covinfected cells. agents being developed to target p mapk and its downstream molecules (for downregulation) and akt (for upregulation) may be important for the design of anti-sars-cov drugs. murine coronavirus replication-induced p mitogen-activated protein kinase activation promotes interleukin- production and virus replication in cultured cells regulation of cell death protease caspase- by phosphorylation mammalian map kinase signalling cascades induction of il- release in lung cells via activator protein- by recombinant baculovirus displaying severe acute respiratory syndrome-coronavirus spike proteins: identification of two functional regions cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis stat serine phosphorylation by erk-dependent and -independent pathways negatively modulates its tyrosine phosphorylation high-dose hydrocortisone reduces expression of the pro-inflammatory chemokines cxcl and cxcl in sars coronavirus-infected intestinal cells inhibition of glycogen synthase kinase- by insulin mediated by protein kinase b akt phosphorylation of bad couples survival signals to the cell-intrinsic death machinery mapk pathways in radiation responses characterization of a unique groupspecific protein (u ) of the severe acute respiratory syndrome coronavirus hemopoietic growth factors with the exception of interleukin- activate the p mitogen-activated protein kinase pathway interleukin- activates a novel protein kinase cascade that results in the phosphorylation of hsp hsp and hsp : potentially oncogenic apoptosis inhibitors organization and regulation of mitogenactivated protein kinase signaling pathways eif initiation factors: effectors of mrna recruitment to ribosomes and regulators of translation constitutive activation of stat signaling abrogates apoptosis in squamous cell carcinogenesis in vivo activation of ap- signal transduction pathway by sars coronavirus nucleocapsid protein an interferon-gamma-related cytokine storm in sars patients creb and its associated proteins act as survival factors for human melanoma cells molecular cloning of rat rac protein kinase alpha and beta and their association with protein kinase c zeta ngf rescues human b lymphocytes from anti-igm induced apoptosis by activation of pkc mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation altered p mitogen-activated protein kinase expression in different leukocytes with increment of immunosuppressive mediators in patients with severe acute respiratory syndrome microarray and real-time rt-pcr analyses of differential human gene expression patterns induced by severe acute respiratory syndrome (sars) coronavirus infection of vero cells the genome sequence of the sars-associated coronavirus tyrosine dephosphorylation of stat in sars coronavirus-infected vero e cells phosphorylation of p mapk and its downstream targets in sars coronavirus-infected cells importance of akt signaling pathway for apoptosis in sars-cov-infected vero e cells jnk and pi k/akt signaling pathways are required for establishing persistent sars-cov-infection in vero e cells constitutive activation of stat in human prostate tumors and cell lines: direct inhibition of stat signaling induces apoptosis of prostate cancer cells protein kinase c-subspecies from brain: its structure, expression and properties multiple routes to astrocytic differentiation in the cns characterization of a novel coronavirus associated with severe acute respiratory syndrome the nucleocapsid protein of the sars coronavirus is capable of self-association through a c-terminal amino acid interaction domain the sars coronavirus nucleocapsid (n) protein induces actin reorganization and apoptosis in cos- cells overexpression of a, a protein specifically encoded by the severe acute respiratory syndrome coronavirus, induces apoptosis via a caspase-dependent pathway dynamic changes in clinical features and cytokine/chemokine responses in sars patients treated with interferon alfacon- plus corticosteroids protein kinase b and rac are activated in parallel within a phosphatidylinositide oh-kinasecontrolled signaling pathway a central control for cell growth plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome sars coronavirus induces apoptosis in vero e cells subcellular localization and membrane association of sars-cov a protein i thank drs. s. fukushi, m. saijo, m. ogata, k. sakai, i. kurane, and s. morikawa (national institute of infectious diseases, japan) for useful suggestions. we acknowledge funding from a grant-in-aid for scientific research from the ministry of education, science, sports and culture of japan, a grantin-aid from the ministry of health, labor, and welfare of japan, and the japan, health science foundation, tokyo, japan. key: cord- - naxn authors: an, hongliu; cai, zhichao; yang, yuying; wang, zhaoxiong; liu, ding xiang; fang, shouguo title: identification and formation mechanism of a novel noncoding rna produced by avian infectious bronchitis virus date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: naxn viral noncoding (nc) rnas have been shown to play important roles in viral life cycle. many viruses employ different mechanism to produce ncrnas. here, we report that coronavirus infectious bronchitis virus (ibv) produces a novel ncrna in virus-infected cells. this ncrna consists of nucleotides excluding a poly(a) tail, is mainly derived from the ′-untranslated region of ibv genome, and contains a -nt-long of terminal leader sequence derived from the ′ end of the viral genome. using mutagenesis and reverse genetics, we reveal that this ncrna is a subgenomic rna generated by discontinuous transcription mechanism. viruses employ different mechanisms to produce a number of noncoding (nc) rnas excluding microrna. these ncrnas mainly include: ( ) viral ncrnas transcribed by rna polymerase (pol) iii. for example, two virus-associated (va) rnas encoded by human adenovirus (reich et al., ; steitz et al., ) , eber and eber encoded by epstein-barr virus (skalsky and cullen, ) , mirna precursors encoded by murine γ-herpesviral trna-pre-mirna chimeras (bogerd et al., ; bowden et al., ; diebel et al., ) and retrovirus (kincaid et al., ) , and intragenic viral small ncrna encoded by human bocavirus (wang et al., ) ; ( ) viral ncrnas transcribed by rna polymerase ii. for example, polyadenylated nuclear (pan) rna encoded by kaposi's sarcoma-associated herpesvirus (sun et al., ; zhong and ganem, ) , the~ -kb latency-associated transcript (lat) expressed by herpes simplex virus (bloom, ), a con-served~ -kb intron expressed by human cytomegalovirus (hcmv) (kulesza and shenk, ) , and a . -kb rna expressed by mouse cmv (kulesza and shenk, ) , u-rich rnas (hsurs) produced by herpesvirus saimiri (hvs), (albrecht and fleckenstein, ; ensser and fleckenstein, ) ; ( ) subgenomic ncrnas from single-stranded rna viruses by incomplete degradation of genomic rna by the cellular - ′ exonuclease xrn . for example, subgenomic rna (sfrna) produced by flaviviruses, such as dengue virus (denv), west nile virus (wnv), yellow fever virus (yfv), and zika virus [reviewed in (bidet and garcia-blanco, ; roby et al., ; pijlman et al., ; akiyama et al., ) , and subgenomic ncrna generated by some plant viruses such as barley yellow dwarf virus and red clover necrotic mosaic virus using similar mechanism (miller et al., a) . viral ncrnas play important roles in viral life cycle. adenovirus va rnas are characterized for their role in counteracting the host antiviral defense through inhibition of protein kinase r (pkr) (mathews and shenk, ; wilson et al., ) . eber regulates the expression of a subset of ebv latent genes throung the interaction of eber and a cellular transcription factor paired box protein (pax ) (arvey et al., ; lee et al., ) . degradation of mir- by mediated hsur promotes activation and presumably proliferation of hvs-infected host t cells guo et al., ) . kshv pan rna is essential for virion production (borah et al., ) .β-herpesvirus hcmvencoded β . prevents mitochondria-induced apoptosis, enabling steady atp production for viral processes and persistent infection (campbell et al., ; stern-ginossar et al., ) . sfrna produced by flaviviruses is required for cytopathicity and pathogenicity (pijlman et al., ) . it has been demonstrated to ) interfere with cellular rna decay pathways by inhibiting xrn (moon et al., ) , ) dampen the antiviral activity of type i interferon (schuessler et al., ) and ) inhibit the rnai pathway in both the vertebrate and arthropod hosts, most likely by serving as a decoy substrate for dicer (schnettler et al., ) . inhibition of the host interferon response appears to be, at least in some flaviviruses, achieved by binding and inactivating cellular https://doi.org/ . /j.virol. . . received september ; received in revised form december ; accepted december regulators of translation of interferon-upregulated mrnas (bidet et al., ) . ncrnas encoded by some plant rna viruses can inhibit host translation and overwhelm host's rna interference system to favor virus infection (miller et al., b) . avian infectious bronchitis virus (ibv) belongs to the genus gammacoronavirus within the order nidovirales. ibv is an enveloped positive-sense, single-stranded rna virus causing the acute highly contagious poultry disease infectious bronchitis (cavanagh, ) . like other coronaviruses, ibv can produce sgrnas via a discontinuous transcription mechanism to encode its structural proteins and specific accessory proteins (masters, ; sawicki et al., ) . briefly, ibv produces six mrna species in the infected cells, including its genomic mrna , sgrna encoding spike (s) protein, sgrna encoding a, b and envelope (e) protein, sgrna encoding membrane (m) protein, sgrna encoding a and b, and sgrna encoding nucleocapsid (n) protein. recently, a low-abundance sgrna located between the sgrna and has been identified (bentley et al., ) . in this study, we identify firstly an ncrna in the ibv-infected cells. moreover, we prove that this ncrna is derived mainly from ′ utr of viral genome and is generated by discontinuous transcription process. vero and chicken embryo fibroblast df cells were maintained in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs), penicillin ( units/ml) and streptomycin ( units/ml) (invitrogen). a recombinant ibv (ribv) (fang et al., ) generated from an infectious clone reference genome (ibv beaudette p , genbank accession number dq . ) was used as the wild-type control. all ibv mutants were propagated in vero cells in fbs-free dmem. virus stocks were made through three repeated freezethaw cycles and kept at − °c in . - ml aliquots until use. constructs containing mutation or deletion were produced by using a quikchange site-directed mutagenesis kit (stratagene). the fulllength cdna was assembled as previously described (fang et al., ) by replacing the corresponding fragment with the mutant fragment. the mutations were verified by automated nucleotide sequencing. fulllength transcripts were generated in vitro using the mmessage mmachine t kit (ambion, austin, tx) according to the manufacturer's instructions, and electroporated into vero cells with one pulse at v and µf with a bio-rad gene pulser ii electroporator. the transfected vero cells were cultured overnight in % fbs-containing dmem and further cultured in dmem without fbs. the transfected cells were monitored daily for formation of cytopathic effect (cpe). recovered viruses were plaque purified and passaged on vero cells. vero cells in -well plates were infected with a dilution series of viruses for h, washed twice with medium. cells were overlaid with . % agar in fbs-free dmem, incubated at ℃ for - days, fixed with % formaldehyde, and stained with . % crystal violet. the number of plaques was counted and the virus titer was calculated as plaque-forming unit (pfu) per ml. ten-day-old embryonated, pathogen-free chicken eggs were inoculated with ibv as described previously (shen et al., ) . the allantoic fluid and different organs were harvested after the embryos were chilled at °c overnight. total rna was extracted from the homogenized tissues and used for rt-pcr analysis. total rna was isolated from ibv-infected cells using trizol reagent® (invitrogen) according to the manufacturer's instructions. the concentration of the total rna extracted was quantified using a nanodrop™ spectrophotometer (nanodrop technologies, inc., thermo fisher scientific, usa). reverse transcription (rt) was performed with oligo(dt) or specific primer using reverse transcriptase (promega) according to the manufacturer's instructions. for the detection of viral positive-stranded subgenomic (sg)rna, oligo(dt) was used for cdna synthesis; for negative-stranded sgrna, ibv- ′end-f ( ′- acttaagatagatattaatatata) was used. ibv- ′end-f and ibv- ′end-r ( ′- tgctctaactctatactagc) were used for pcr. ibv-infected cells at different time point post-infection were washed with pbs and lysed with ×sds loading buffer containing mm dithiothreitol (dtt), boiled at °c for min, and clarified. the proteins were separated by sds-page and transferred to a polyvinylidene difluoride (pvdf) membrane (stratagene). the membrane was blocked overnight at °c or for h at room temperature in blocking buffer ( % fat-free milk powder in phosphate-buffered saline (pbs) buffer containing . % tween (pbst)) and then was incubated with diluted primary antibodies in blocking buffer for h at room temperature. after the membrane was washed three times with pbst, it was incubated with : diluted anti-mouse or anti-rabbit igg antibodies conjugated with horseradish peroxidase (dako) in blocking buffer for h at room temperature. after the membrane was washed three times with pbst, the polypeptides were detected with a chemiluminescence detection kit (ecl kit; amersham biosciences) according to the manufacturer's instructions. the films were exposed and developed. quantitative real-time pcr (qpcr) was used to validate gene expression changes in infected cells. total rna ( µg) was reversedly transcribed to cdna, and the resulting cdna was subjected to qpcr using power sybr green pcr master mix (applied biosystems). amplification and data collection were performed as manufacturer's instruction (applied biosystems real-time pcr system). the relative gene expression levels were measured using gapdh as an internal reference, and normalized to gene expression in mock-infected cells (relative expression = ). all experiments were performed in triplicate. in the virus-infected cells, ibv produces six mrna species, including the genome-length mrna and five subgenomic mrnas via a discontinuous transcription mechanism. this mechanism is mediated by transcription-regulating sequences (trss) in the ′ end of the leader (trs-l) and the preceding each mrna body (trs-b). trss comprise a conserved core sequence (cs) [cu(u/g)aacaa] in ibv. each mrna possesses a leader sequence of nucleotides derived from the ′-end of the genome. in general, six mrnas are readily detected by northern blotting in the infected cells. when probing the viral positive-or negative-stranded mrna by rt-pcr, an unexpected pcr product was concurrently amplified in the ibv-infected vero cells as well as in chicken embryos (fig. ) . it is smaller than mrna . subsequently, this product was cloned and sequenced. sequence analysis indicated that it consists of nucleotides excluding a poly(a) tail, is mainly derived from the ′-utr (from nucleotides - ) of ibv genome, and contains a -nt-long of terminal leader sequence derived from the ′ end of the viral genome (fig. ) . the results showed the generation of a novel sgrna in the ibvinfected cells. this sgrna may be overlooked in previous studies because of its smaller size and lower level of transcription. due to lack of start codon aug, this sgrna is designated as a noncoding rna (ncrna). insertion of an orf encoding egfp (carrying its own start codon) between and nt of ibv ′-utr allowed the recovery of recombinant virus. rt-pcr analysis showed the presence of the egfp-containing sgrna and fluorescence confirmed egfp expression in virus-infected cells (fig. ) , implying that the ncrna is an mrna. however, this virus was unstable and a deletion of nucleotide acids (from to nt) of egep sequences was detected in passage in vero cells (data not shown). further sequence comparison revealed that a sequence motif (uaaca), located in the junction between the ′ end leader sequence and the ′-utr of ncrna, is shared by ′-terminus of ′-utr and the core sequence (cuuaacaa) within ibv trs (fig. ) . these results prompt us to speculate that this ncrna, like the other viral sgrna, may be generated by a discontinuous transcription mechanism via template switch mediated by a noncanonical core sequence (uaaca). to confirm this hypothesis, we analyzed the effect of several mutations on ncrna generation by mutagenesis and reverse genetics. as shown in fig. , compared to wild-type ribv, single mutation u a and a u had no or minor effect on ncrna synthesis; a u resulted in a significant reduction in the ncrna generation, while mutation c g, a u and deletion of five nucleotides (Δ - ) completely abolished the synthesis of both positive-stranded and negative-stranded ncrna (fig. ) , suggesting that at least three fig. . detection of a novel sgrna in virus-infected vero cells and chicken embryo by rt-pcr. total rna was extracted from the ribv-infected vero cells and chicken embryo. cdna was synthesized by reverse transcription using oligo (dt) as a primer. pcr was performed using primers ibv- ′end-f and ibv- ′end-r. the amplicons were analyzed on % agarose gel electrophoresis. fig. . sequence of the junction between ibv ′-utr and the leader at ′-end of viral genome, indicating the formation of a novel sgrna mediated by uaaca . vero cells were infected with ribv at an moi of pfu/cell. total rna extracted from the infected cells was used for reverse transcription using primer oligo(dt) . pcr was carried out using primers ibv- ′end-f and ibv- ′end-r. pcr product was cloned and sequenced. nucleotides (a /a /c ) are involved in the effective ncrna generation. the results confirmed previous report that ibv can synthesize sgrna via template switch mediated by a noncanonical core sequence (bentley et al., ) notably, the mutant virus carrying four mutations (a u/a u/g c/g c) was also unable to produce ncrna (fig. ) , suggesting these nucleotides are required for ncrna production. because the sequence motif (a /a / g /g ) located downstream of the truncated cs (uaaca) also exists downstream of the cs (cuuaacaa) in the leader trs (table , marked in bold), our result prove that the sequences downstream of the cs exert a stronger influence on coronavirus sgrna synthesis (sola et al., ) . taken together, the results confirm that the ncrna generation involves a discontinuous transcription process in which the ′ leader sequence and ′-utr are fused through the transcription-regulating sequences in the ′ end of the leader and in the ′ end of the ′-utr. the mutant virus c g was selected for further experiments because it is not capable of producing ncrna and carries only one nucleotide change, compared to ribv. plaque assay on vero cells showed both viruses did not display major differences in plaque morphology and in virus growth properties (fig. a) . moreover, real-time rt-pcr and western blot were performed to analyze the expression of s and n genes at different time points post-infection, respectively. similarly, no significant differences were detected at both rna level and protein level (fig. b, c) . the results suggest that ncrna has little effects on viral replication and viral cytopathicity in vero cells. in this report, we have identified an ncrna in ibv-infected cells and revealed that this ncrna is generated via discontinuous transcription mechanism by reverse genetics and mutagenesis for the first time. although the ncrna has no or little effect on viral replication and pathogenesis in vero cells, roles in ibv pathogenesis in chicken and virus-host interplay are unknown, needing to further study. coronaviruses employ a discontinuous transcription mechanism to synthesize subgenomic mrnas through template switch taking place in the transcription-regulating sequences in the ′ end of the leader (trs-l) and in the intergenic region preceding each mrna body (trs-b) during negative rna synthesis (baric and yount, ; zúñiga et al., ; masters, ; sawicki et al., ) . the five sgrnas (mrnas - ) of ibv, which are readily detected by northern blotting, possess the canonical core sequence ( ′-cu(u/g)aacaa- ′) ( table ). it has been reported that coronaviruses, such as severe acute respiratory syndrome coronavirus, mouse hepatitis virus and ibv, can use noncanonical cs to synthesize sgrna via discontinuous transcription mechanism (zhang and liu, ; hussain et al., ; bentley et al., ) . in this report, we have identified the existence of a novel sgrna derived mainly from the ′ utr of ibv in the ibv-infected cells (fig. ) . the synthesis of this sgrna is mediated by a truncated cs ( uaaca ) identical to nucleotides of - of ibv cs. among which at least three nucleotides (a /a /c ) are involved in the effective sgrna generation (figs. and ) , providing more evidence for the use of noncanonical transcriptional signals in synthesis of coronavirus sgrnas. in addition, we verified that the sequence motif (a /a /g /g ) located downstream of the truncated cs (uaaca) is necessary for ncrna generation (fig. ) , reinforcing the importance of nucleotides immediately flanking cs in coronavirus sgrna synthesis (sola et al., ) . interestingly, when blast search in genbank, we find that the sequence motif (uaaca) is conserved at ′ end of ′ utr of ibv strain beaudette and its derivants, arkansas dp , and turkey coronavirus but not for strain m , h , h , a , and several field isolates in china. therefore, whether these viruses can produce ncrna and how ncrna affects the viral pathogenecity remain to be determined. fig. . effect of ncrna on plaque morphology and viral replication. a. plaque assay. vero cells in -well plates were infected with a dilution series of ribv and mutant virus ibv-c g for h, respectively. after washing twice with medium, cells were overlaid with . % agar in fbs-free dmem, incubated at ℃ for - days, fixed with % formaldehyde, and stained with . % crystal violet. b. quantitative analysis of sgrna synthesis of n and s. total rna ( μg) extracted from the vero cells infected with ribv and ibv-c g at an moi of . pfu/cell at and h post-infection 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virus replication dissection of the adenoviral va rnai central domain structure reveals minimum requirements for rna mediated inhibition of pkr identification of a noncanonical signal for transcription of a novel subgenomic mrna of mouse hepatitis virus: implication for the mechanism of coronavirus rna transcription characterization of ribonucleoprotein complexes containing an abundant polyadenylated nuclear rna encoded by kaposi's sarcoma-associated herpesvirus (human herpesvirus ) sequence motifs involved in the regulation of discontinuous coronavirus subgenomic rna synthesis this work was financially supported by grants from the national natural science foundation of china (no. ) and the department of science and technology, hubei provincial people's government, china (no. bhe ) key: cord- -cz t n authors: jansen van vuren, petrus; wiley, michael; palacios, gustavo; storm, nadia; mcculloch, stewart; markotter, wanda; birkhead, monica; kemp, alan; paweska, janusz t. title: isolation of a novel fusogenic orthoreovirus from eucampsipoda africana bat flies in south africa date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: cz t n we report on the isolation of a novel fusogenic orthoreovirus from bat flies (eucampsipoda africana) associated with egyptian fruit bats (rousettus aegyptiacus) collected in south africa. complete sequences of the ten dsrna genome segments of the virus, tentatively named mahlapitsi virus (mahlv), were determined. phylogenetic analysis places this virus into a distinct clade with baboon orthoreovirus, bush viper reovirus and the bat-associated broome virus. all genome segments of mahlv contain a ' terminal sequence ( '-gguca) that is unique to all currently described viruses of the genus. the smallest genome segment is bicistronic encoding for a kda protein similar to p membrane fusion protein of bush viper reovirus and an kda protein similar to p non-structural protein of baboon orthoreovirus. this is the first report on isolation of an orthoreovirus from an arthropod host associated with bats, and phylogenetic and sequence data suggests that mahlv constitutes a new species within the orthoreovirus genus. bats have been increasingly associated with emerging and re-emerging viruses. the likelihood of possible transmission of these pathogens to humans is ever increasing as a result of human encroachment on animal habitats, climate change and change of human behaviour. pathogens of particular public health importance are filoviruses [ , ] , coronaviruses [ , ] , paramyxoviruses [ , ] and lyssaviruses [ , ] . other viruses, without a known human disease link, have also been detected recently [ ] [ ] [ ] . some human pathogens, such as rift valley fever virus, that have been detected in bats were likely a result of coincidental infection and do not constitute proof that bats play a role as reservoirs [ ] . bats are parasitized by a number of ectoparasites, including mites, bat flies, ticks and fleas, and often by some or all of these simultaneously [ ] . the bat flies are members of two families in the diptera order, namely, the streblidae and nycteribiidae, and are highly host-specific obligate ectoparasites of bats [ ] [ ] [ ] . both bat fly families are hematophagous and potentially capable of (qiagen). cellular debris was removed by centrifugation at , ˆg for min, and the supernatant used for subsequent virus isolation and nucleic acid extraction procedures. viruses , , x mm stainless steel beads (qiagen). cellular debris was removed by centrifugation at , × g for min, and the supernatant used for subsequent virus isolation and nucleic acid extraction procedures. the wells of -well tissue culture plates (nunc) were seeded with vero e cells and grown to %- % confluency in eagle's minimum essential medium (emem, lonza) supplemented with antibiotics (penicillin/streptomycin/amphotericinb, lonza) and % foetal calf serum at °c and % co . culture medium was removed and the monolayers in individual wells inoculated with μl of ectoparasite pool homogenates (one pool representing parasites from one bat). after one hour adsorption at °c, the inoculum was removed and fresh emem containing antibiotics and % foetal calf serum added. the -well plates were incubated for days and cytopathic effects (cpe) monitored. a second and third blind passage was performed for all samples by inoculating and incubating monolayers as described above with μl of undiluted supernatant from the preceding passage. supernatants were collected from all wells displaying cpe after three blind passages, a / dilution prepared in emem, and ml of this used to inoculate a cm tissue culture flask. if the same cpe was noted in the sub-cultured cm flask, a / dilution of this supernatant was prepared and used to inoculate a cm tissue culture flask for preparation of stock virus. stock virus titres were determined by standard tissue culture infectious dose (tcid ) titrations on -well microtitre plates as described previously [ ] . the wells of -well tissue culture plates (nunc) were seeded with vero e cells and grown to %- % confluency in eagle's minimum essential medium (emem, lonza) supplemented with antibiotics (penicillin/streptomycin/amphotericinb, lonza) and % foetal calf serum at ˝c and % co . culture medium was removed and the monolayers in individual wells inoculated with µl of ectoparasite pool homogenates (one pool representing parasites from one bat). after one hour adsorption at ˝c, the inoculum was removed and fresh emem containing antibiotics and % foetal calf serum added. the -well plates were incubated for days and cytopathic effects (cpe) monitored. a second and third blind passage was performed for all samples by inoculating and incubating monolayers as described above with µl of undiluted supernatant from the preceding passage. supernatants were collected from all wells displaying cpe after three blind passages, a / dilution prepared in emem, and ml of this used to inoculate a cm tissue culture flask. if the same cpe was noted in the sub-cultured cm flask, a / dilution of this supernatant was prepared and used to inoculate a cm tissue culture flask for preparation of stock virus. stock virus titres were determined by standard tissue culture infectious dose (tcid ) titrations on -well microtitre plates as described previously [ ] . for electron microscopy specimen preparation, %- % confluent vero e monolayers in cm flasks were inoculated with stock virus and monitored for cpe. at the first sign of cpe, culture supernatant was collected, cleared of cellular content by centrifugation ( ˆg for min), and subsequently fixed in an equal volume of . % glutaraldehyde in . m hepes buffer (ph . ) for visualization of virus particles by negative staining. a beckman airfuge ® (beckman coulter, brea, ca, usa) was used to concentrate all samples ( min at kpa), after which droplets of sample were adsorbed to . % formar-coated copper grids for a minimum of min, rinsed twice in deionised, distilled water and stained briefly in % phosphotungstic acid (ph . ). for ultramicrotomy, the remaining infected monolayers were flooded with the same fixative overnight, then routinely processed (postfixation in % buffered osmium tetroxide, graded ethanol dehydration, infiltration with a low viscosity resin (agar scientific, stansted, uk) and overnight polymerisation at ˝c). seventy nm sections were cut on a leica em-uc , double stained with saturated uranyl acetate and lead citrate, and viewed at kv on a biotwin spirit (fei company, hillsboro, or, usa). imaging was done with an olympus quemesa ccd camera (olympus, tokyo, japan). single-primer amplification (sispa), rapid amplification of cdna ends (race), next-generation sequencing (ngs) and bioinformatics stock virus culture supernatant was added to trizol-ls (life technologies, waltham, ma, usa) at a ratio of µl supernatant to µl trizol-ls. rna was extracted using a column based kit (direct-zol rna kit, zymo research, irvine, ca, usa). to increase sensitivity, rrna was depleted using the same method as described previously [ ] . rnas were converted to cdna and amplified using sispa as described previously with modifications [ ] . to enhance coverage of the terminal ends, an oligo containing three rgtp at the ' end (gccggagctctgcagatatcggccattat ggccrgrgrg) was added during first-strand cdna synthesis and the reverse transcriptase was changed to maxima h minus (thermo scientific, waltham, ma, usa), which has terminal transferase activity that enables addition of the rgtp containing oligo to the ' end during cdna synthesis. amplicons were sheared and libraries prepared using the illumina truseq dna library preparation kit (illumina, san diego, ca, usa). sequencing was performed either on an illumina miseq (illumina) or nextseq (illumina) using either a ˆ or ˆ version kit. illumina and sispa adapter sequences were trimmed from the sequencing reads using cutadapt- . . [ ] , quality filtering was conducted with prinseq-lite (-min len -derep -lc method dust-lc threshold -trim ns left -trim ns right -trim qual right ) [ ] and reads were assembled into contigs using ray meta with kmer length = [ ] . resultant contigs were aligned to the ncbi sequence database using blast. the mega (version ) program was used to prepare alignments (clustalw) of nucleic acid segment sequences, deduced amino acid sequences, phylogenetic trees and pairwise distance calculations [ ] . the publicly available reovirus sequences used in the analysis were obtained from ncbi-nucleotide (genbank). nucleotide sequences from a small number of viruses from each genus in the reoviridae family were used to prepare a maximum likelihood tree showing the placement of mahlv in the family based on the full rna-dependent rna polymerase (rdrp) encoding segment. maximum likelihood trees were prepared using amino acid sequences of all open reading frames from all segments, showing the placement of mahlapitsi virus (mahlv) in the orthoreovirus genus relative to other viruses in this genus for which sequence is available on genbank. virus sequence accession numbers are summarised in table . the evolutionary history was inferred by using the maximum likelihood method based on the jtt matrix-based model [ ] . the tree with the highest log likelihood is shown. the percentage of trees in which the associated taxa clustered together is shown next to the branches ( bootstrap iterations). initial tree(s) for the heuristic search were obtained by applying the neighbor-joining method to a matrix of pairwise distances estimated using a jtt model. the tree is drawn to scale, with branch lengths measured in the number of substitutions per site all positions containing gaps and missing data were eliminated. evolutionary analyses were conducted in mega [ ] . open reading frames were located and deduced protein amino acid sequences prepared by using the clc genomics workbench (qiagen). putative functions of the new virus deduced proteins were determined by blastx similarity searches to sequences available on genbank. the ectoparasite pool homogenate used for virus isolation was used as dna source for phylogenetic confirmation of species. dna was extracted using trizol (invitrogen, waltham, ma, usa) and the method as described by the manufacturer. amplification of the cytochrome c oxidase subunit i (coi) gene was performed with barcoding primers as described by tortosa et al.: lco and hco [ ] . polymerase chain reaction was carried out in µl reactions containing µl mytaq red mix ˆ(bioline, london, uk), µl of forward and reverse primer ( µm), dna template ( µl) and nuclease free water ( µl). amplification steps were ˝c for min, cycles of ˝c for s, ˝c for s and ˝c for s, and ˝c for min. pcr product was purified using the minelute kit (qiagen). purified pcr amplicon products were then sequenced at the nicd core sequencing facility (nicd, sandringham, south africa). replication of mahlv was evaluated in two cell lines: vero e (source african green monkey kidney) and c - (source aedes albopictus mosquitoes). cells were grown to %- % confluency in cm flasks, supernatant removed and respective flasks inoculated with ml of ´ , ´ , ´ and ´ dilutions from stock virus ( ˆ tcid /ml) in emem. after h adsorption at ˝c (veroe ) or ˝c (c - ), the inoculum was removed, cells washed with ml phosphate buffered saline (pbs) and fresh emem, antibiotics and % foetal calf serum (hyclone, logan, ut, usa) added. cultures were incubated for days at ˝c (veroe ) or ˝c (c - ) while . ml aliquots of supernatant were collected from each flask directly after inoculation and addition of fresh medium (day ), followed by day , and . rna was extracted from µl of the serial supernatant collections (qiamp viral rna kit, qiagen) and subjected to taqman real-time rt-pcr. a taqman real-time rt-pcr was developed to detect the rdrp gene of mahlv. primers and probe sequences are: forward morv_ f ( '-tagtggttcgtatgcgtggt- '), reverse morv_ r ( '-aacagccattcaatctcagg- ') and probe morv_ p (fam-ggcacatatccctcaactgg-bhq), with the number in the oligonucleotide name indicating the nucleic acid position in the segment encoding rdrp. real-time rt-pcr was performed on the extracted rna using the qiagen one-step rt-pcr kit (qiagen) on a smartcycler (cepheid, sunnyvale, ca, usa) with the following program: reverse transcription ( ˝c for min), hot-start taq activation ( ˝c for min) and cycles of amplification ( ˝c for s; ˝c for s plus signal acquisition; ˝c for s). rna extracted from diluted stock mahlv (final ˆ tcid /ml) was used as a qualitative positive control in each run. from a total of bat ectoparasite pools subjected to virus isolation by three blind passages, two yielded an agent that caused obvious cytopathic effects in the form of syncytia (giant cell) formation by three or four days post inoculation (d.p.i.) (figure ). the parasite pool that yielded mahlv isolate was collected from an apparently healthy adult female rousettus aegyptiacus bat captured at mahune cave in may . cpe in vero cells were noted after two blind passages, and the supernatant collected on day five from passage four in a cm flask containing infected vero cells yielded ˆ . tcid /ml of the unknown virus. the second parasite pool that yielded mahlv isolate - was collected from an apparently healthy juvenile male rousettus aegyptiacus bat captured at mahune cave in june . cpe in vero cells were noted after three blind passages, and the supernatant collected on day five from passage five in a cm flask containing infected vero cells yielded ˆ tcid /ml of the virus. these supernatants, passage four of and passage five of - , were used for subsequent identification by tem and ngs. the ectoparasites from which the viruses were isolated were morphologically identified as bat flies, eucampsipoda africana theodor (diptera: nycteribiidae) ( figure ) [ ] . sequencing of the cytochrome c oxidase subunit i gene (coi) and alignment to sequences available on genbank, followed by phylogenetic analysis (figure ) confirms that the bat flies in this study are closest related to eucampsipoda spp. identified before. initial screening of negatively-stained culture supernatants revealed the presence of rounded icosahedrons lacking envelopes, which resembled non-rotavirus-like virions of the reoviridae ( figure ). two-layered capsids with an outer diameter of - nm (n = ) and an inner core of - nm, possessed clearly defined solvent channels radiating outwards through the clustered capsomers of the outer capsid layer ( figure ). although the dimensions of the negatively-stained, inner capsid layer were comparable to those recorded after processing for ultramicrotomy, the outer layer was slightly larger, occasionally measuring up to nm in diameter. the coi partial sequence from the bat fly sequenced from this study is indicated by the red star. initial screening of negatively-stained culture supernatants revealed the presence of rounded icosahedrons lacking envelopes, which resembled non-rotavirus-like virions of the reoviridae ( figure ). two-layered capsids with an outer diameter of - nm (n = ) and an inner core of - nm, possessed clearly defined solvent channels radiating outwards through the clustered capsomers of the outer capsid layer ( figure ). although the dimensions of the negatively-stained, inner capsid layer were comparable to those recorded after processing for ultramicrotomy, the outer layer was slightly larger, occasionally measuring up to nm in diameter. (a) negatively-stained particle with two distinct layers; (b) icosahedral negatively-stained particle; (c) resin-embedded, sectioned viral particle in cytoplasm of infected vero e cell. white arrows indicate some of the characteristic spaces between the finger-like, capsomeric projections surrounding the solvent channels through the outer capsid layer. evident in ultrathin sections of infected vero e cells were multinucleate cells with extensive nuclear lobing, and cytoplasmic inclusion bodies associated with developing, double-shelled virus particles ( figure ). tem observations therefore suggested that the isolated virus belonged to the reoviridae, sub-family spinareovirinae. (a) negatively-stained particle with two distinct layers; (b) icosahedral negatively-stained particle; (c) resin-embedded, sectioned viral particle in cytoplasm of infected vero e cell. white arrows indicate some of the characteristic spaces between the finger-like, capsomeric projections surrounding the solvent channels through the outer capsid layer. evident in ultrathin sections of infected vero e cells were multinucleate cells with extensive nuclear lobing, and cytoplasmic inclusion bodies associated with developing, double-shelled virus particles ( figure ). tem observations therefore suggested that the isolated virus belonged to the reoviridae, sub-family spinareovirinae. evident in ultrathin sections of infected vero e cells were multinucleate cells with extensive nuclear lobing, and cytoplasmic inclusion bodies associated with developing, double-shelled virus particles ( figure ). tem observations therefore suggested that the isolated virus belonged to the reoviridae, sub-family spinareovirinae. an unbiased next-generation sequencing approach using sispa amplification confirmed the presence of a novel orthoreovirus. initial sequencing results of both isolates yielded enough sequence coverage to identify all segments. a polyetheleneglycol (peg) precipitated preparation of isolate - yielded the most viral specific reads and formed contigs aligning to othoreoviruses using blastn and blastx. both the ' and ' ends were missing for all the segments, so to obtain complete genomes for each isolate, rrna depletion and a combination of sispa and rapid amplification of cdna ends (sispa-race) was done. read numbers were also increased by running samples on an illumina nextseq . both an increase in the percentage of viral reads aligning to the genome segments and an increase in coverage of the ends were observed. presence of mahlv in the original homogenates from which the isolates were obtained was confirmed by sispa amplification and ngs directly from the homogenates. expectedly, only a low number of reads from both homogenates mapped to the mahlv sequence due to the high amount of host sequence obscuring viral specific sequences combined with likely low viral load in the homogenates and the relatively low sensitivity of the sispa method. a maximum likelihood tree, constructed with nucleic acid sequence data for the rna-dependent rna polymerase (rdrp) encoding segments of representative viruses from the different genera within reoviridae (figure ) shows the placement of both isolates amongst other orthoreoviruses in the family. maximum likelihood trees were prepared using the deduced amino acid sequences from the open reading frames (orf's) of all the virus' segments and those of other viruses in the orthoreovirus genus (figures - ) . a distinct clade is formed by mahlv, bush viper reovirus, baboon orthoreovirus and broome virus within the genus. the above-mentioned clade is visibly distinct from others composed of bat-associated viruses; the nelson bay orthoreovirus and bat-derived mammalian orthoreoviruses. the closest relative of mahlv, based on sequence homology of a conserved core protein, is bush viper reovirus (lambda b nucleic acid identity- . %; rdrp amino acid identity- . %) while the closest bat-associated virus is broome virus (lambdab nucleic acid identity- . %; rdrp amino acid identity- . %) ( table ) . homology of the divergent major outer capsid protein of mahlv to known orthoreoviruses is much lower: sigma b nucleic acid identity- . %- . %; amino acid identity- . %- . % (table ) . the genome segments of mahlv were named according to the nucleotide length, which is consistent with the nomenclature of other orthoreoviruses [ ] . a summary of the mahlv genome is given in table . the total genome size is , nucleotides and predicted to encode eleven proteins, seven of which are structural. all ten genome segments of mahlv contain an identical ' terminal sequence, ucauc- ', which is conserved between all known species of orthoreovirus, and an identical ' terminal sequence, '-gguca which is unique to mahlv. non-coding regions (ncrs) are present at both ends of the genome segments, with the ' ncrs being shorter in nucleotide length than ' ncrs. the nucleotide sequences of the two isolates of mahlv, and - , are not identical. nucleotide homology of the rdrp encoding segment between the two isolates is . % ( . % deduced amino acid sequence), and . % ( . % deduced amino acid sequence) for the sigma b encoding segment. putative protein functions were determined by blastx similarity searches to sequences available on genbank, revealing putative functions known for other orthoreoviruses. the segments l , l , m , m , s , s and s each contain a single start aug codon in close proximity to the ' end. the l segment of mahlv contains two aug start codons in close proximity to the ' end, at positions and . agcaugg) . the open reading frame initiating at position is nucleotides in length and putatively encodes for an outer capsid protein involved in membrane penetration during infection (mub). the second aug initiates a nucleotide open reading frame but the deduced amino acid sequence does not match any viral protein of note on genbank. the deduced sigma a protein of mahlv (segment s ) contains the fusogenic orthoreovirus-wide conserved arginine amino acid at position . the s segment is bicistronic and encodes for a kda protein similar to p membrane fusion protein of bush viper reovirus and a non-overlapping kda protein similar to p non-structural protein of baboon orthoreovirus without a known function ( table ). the isoelectric point of mahlv p is acidic ( . ), similar to that of p of broome virus and baboon orthoreovirus and contrary to that of other orthoreoviruses. the first ten amino acids in the putative kda protein of mahlv are identical to the first ten amino acids in the p fusion protein of broome virus and represent the myristoylation consensus sequence required for fusion activity of the protein. the s segment does not encode a cell attachment protein, an observation also characteristic of broome virus and baboon orthoreovirus. . . . . . . . . mahlv replicated efficiently in vero cell culture, with the inoculum containing a high dose of virus ( tcid /ml) leading to rapid monolayer destruction after inoculation, with a peak in virus rna (measured by real-time rt-pcr) by day , followed by a decrease on day . the inoculums containing a lower virus dose ( and tcid /ml) resulted in a peak of rna detection on day . all three above-mentioned inoculum doses yielded detectable virus rna by day after inoculation. the inoculum containing tcid /ml virus did not generate detectable viral rna until day , and was still showing an upward trend on day (last sampling day). the virus did not replicate in the insect cells (c - ) up to day , but adaptation to these cells through serial passaging was not attempted. the role of bats in harbouring pathogens of public health and veterinary importance is becoming an increasingly popular topic of research within the field of emerging and zoonotic diseases. the most notable viruses in which natural transmission from bats have been implicated include filoviruses, coronaviruses, paramyxoviruses, herpesviruses, lyssaviruses and bunyaviruses. the implication of bats in transmission or maintenance of some of these viruses is very circumstantial and often based only on serological evidence. more convincing evidence for others is based on detection of viral nucleic acid and isolation of live virus, although this does not conclusively prove that a vertebrate host is a reservoir. finding pathogens in bats leads to the questions of how they are transmitted between bats, and from bats to incidental hosts such as humans. one possible transmission mechanism could be bat-associated hematophagous arthropods, such as the bat flies, but migration of parasites between bats is not well understood and would require further entomological investigation to better understand [ ] . two isolates of a novel fusogenic orthoreovirus were cultured and we determined their full genome sequences, which were compared to currently known viruses in the genus. the two isolates are not identical but similar enough to suggest that they are merely two isolates of the same virus. this suggests that there are multiple variants of the virus present in the host population. members of a species within the orthoreovirus genus are usually identified by a number of characteristics: amino acid and nucleotide sequence identity, organization of the polycistronic genome segment and host species [ ] . for conserved core proteins, an amino acid identity > % for homologous proteins indicates that two viruses belong to the same species, while identity < % indicates a possible new species. when comparing the amino acid sequence of more divergent outer capsid proteins, > % identity indicates one species and < % indicates different species. nucleic acid sequence identity of homologous segments of > % indicates the same species and < % a new species. the nature of conserved genome segment termini sequences of orthoreoviruses is also useful for virus classification [ ] . the divergence of mahlv sequence from other known orthoreoviruses combined with a unique conserved ' genome segment end and a unique host species, suggests that this is a new virus species in this genus. along with broome virus and baboon orthoreovirus, mahlv is the third orthoreovirus that lacks an identified cell attachment protein in the s segment. this unique characteristic further strengthens the phylogenetic classification which places these viruses in a separate clade and suggests that entry of these viruses into cells is mediated differently than for other orthoreoviruses. taking the abovementioned criteria and the sequence characteristics of the novel virus described here into consideration, we propose that mahlapitsi virus constitutes a new species within the orthoreovirus genus. to our knowledge this is the first description of an orthoreovirus in africa with an indirect link to bats. considering the rich diversity of bat species found on the continent and increased scientific interest in this field, this is unlikely to be the only such virus to be isolated from bat ectoparasites in years to come. however, to our knowledge this is the first orthoreovirus to be isolated from an arthropod host, since all currently known viruses in this genus are associated with vertebrates. mahlv did not replicate on c - cells in this study but aedes albopictus, from which the c - cell line is derived, is classified in a completely different dipteran family, the culicidae, likely pointing to a cell receptor incompatibility. another possible explanation could be the temperature at which insect cells are cultured compared to mammalian cells, which might be incompatible with this virus. the arthropod-borne nature of mahlv transmission needs further investigation, especially to establish whether nycteribiid flies are only involved in mechanical or possibly biological transmission, and if the virus is even transmitted to bats. various other genera in the reoviridae family contain vector-borne viruses, including banna virus (seadornavirus), colorado tick fever virus (coltivirus) and bluetongue virus (orbivirus). our isolation of mahlv from arthropods might direct some attention to the possible role of insects in the transmission of currently known orthoreoviruses, or possibly the presence of other yet unknown viruses in various arthropods. we have no information on the geographical range of mahlv, but the wide distribution of rousettus aegyptiacus in africa and the middle east [ ] , and the strict host preference and specificity of bat flies [ ] [ ] [ ] , dictate that their ranges will overlap. we have no data to suggest that mahlv has any human health implication, but this warrants further investigation. the virus grows to high titers in vero e cells which suggests that it may infect vertebrates, although growth in in vitro systems cannot be translated directly into replication in a vertebrate host. it is important to note, also, that this was after blind passage and cell culture adaptation (cpe noted after two-three blind passages). any risk of human infection for now, however, is only likely in individuals who come into close contact with wild egyptian fruit bats and their ectoparasites. although highly host-dependent, the bat flies have been noted to leave their bat hosts and crawl on bat researchers (personal observation). respiratory disease has been noted in humans infected with melaka, kampar and nelson bay orthoreovirus, including limited human-to-human transmission [ , , , ] . thus the potential for mahlv to infect humans, and spread between humans, cannot be excluded until further investigation is done, especially considering that the virus grows very efficiently on a monkey-derived 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europe reptilian reovirus: a new fusogenic orthoreovirus species investigation of a potential zoonotic transmission of orthoreovirus associated with acute influenza-like illness in an adult patient imported case of acute respiratory tract infection associated with a member of species nelson bay orthoreovirus standards for sequencing viral genomes in the era of high-throughput sequencing isolation of genetically diverse marburg viruses from egyptian fruit bats virological and serological findings in rousettus aegyptiacus experimentally inoculated with vero cells-adapted hogan strain of marburg virus selective depletion of rrna enables whole transcriptome profiling of archival fixed tissue viral genome sequencing by random priming methods cutadapt removes adapter sequences from high-throughput sequencing reads quality control and preprocessing of metagenomics datasets scalable de novo metagenome assembly and profiling molecular evolutionary genetics analysis version . the rapid generation of mutation data matrices from protein sequences an illustrated catalogue of the rothschild collection of nycteribiidae (diptera) in the british museum (natural history), with keys and short descriptions for the identification of subfamilies, genera, species and subspecies sequence at both termini of the genes of reovirus serotype (strain dearing) mapping the zoonotic niche of marburg virus disease in africa the authors would like to thank the following individuals for their contributions towards fieldwork and technical assistance: busi mogodi, justice kgatitsoe, antoinette grobbelaar, terence scott, joe kgaladi, marinda mortlock, marike geldenhuys, jessica coetzer, andre coetzer. we would like to thank dorothy southern and alfred musekiwa for proofreading the manuscript.the project is jointly funded by the following grants awarded to: janusz t. paweska the grant holders acknowledge that opinions, findings and conclusions or recommendations expressed in any publication generated by gdd and nrf-supported research are those of the authors and that the gdd and nrf accept no liability whatsoever in this regard.author contributions: petrus jansen van vuren and janusz paweska conceived and designed the experiments; all authors were involved in some aspect of performing the experiments and interpretation of the data; petrus jansen van vuren, janusz paweska, monica birkhead, michael wiley and gustavo palacios analysed the data; petrus jansen van vuren wrote the paper with inputs from all other authors; janusz paweska, petrus jansen van vuren, wanda markotter and gustavo palacios contributed funding. all authors read and approved the final manuscript. the authors declare no conflict of interest.viruses , , key: cord- -y rw q authors: ogando, natacha s.; dalebout, tim j.; zevenhoven-dobbe, jessika c.; limpens, ronald w.a.l.; van der meer, yvonne; caly, leon; druce, julian; de vries, jutte j. c.; kikkert, marjolein; bárcena, montserrat; sidorov, igor; snijder, eric j. title: sars-coronavirus- replication in vero e cells: replication kinetics, rapid adaptation and cytopathology date: - - journal: j gen virol doi: . /jgv. . sha: doc_id: cord_uid: y rw q the sudden emergence of severe acute respiratory syndrome coronavirus (sars-cov- ) at the end of from the chinese province of hubei and its subsequent pandemic spread highlight the importance of understanding the full molecular details of coronavirus infection and pathogenesis. here, we compared a variety of replication features of sars-cov- and sars-cov and analysed the cytopathology caused by the two closely related viruses in the commonly used vero e cell line. compared to sars-cov, sars-cov- generated higher levels of intracellular viral rna, but strikingly about -fold less infectious viral progeny was recovered from the culture medium. immunofluorescence microscopy of sars-cov- -infected cells established extensive cross-reactivity of antisera previously raised against a variety of non-structural proteins, membrane and nucleocapsid protein of sars-cov. electron microscopy revealed that the ultrastructural changes induced by the two sars viruses are very similar and occur within comparable time frames after infection. furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (remdesivir, alisporivir and chloroquine) is very similar, but that sars-cov- infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha. an important difference between the two viruses is the fact that – upon passaging in vero e cells – sars-cov- apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. these mutations change or delete a putative furin-like cleavage site in the region connecting the s and s domains and result in a very prominent phenotypic change in plaque assays. introduction for the first time in a century, societies and economies worldwide have come to a near-complete standstill due to a pandemic outbreak of a single rna virus. this virus, the severe acute respiratory syndrome coronavirus (sars-cov- ) [ ] belongs to the coronavirus (cov) family, which is thought to have given rise to zoonotic introductions on multiple occasions during the past centuries. coronaviruses are abundantly present in mammalian reservoir species, including bats [ ] , and should now be recognized definitively as a continuous zoonotic threat with the ability to cause severe human disease and explosive pandemic transmission. to date, seven covs that can infect humans have been identified, which segregate into two classes. on the one hand, there are four endemic human covs (hcovs), the first of which were identified in the s, annually causing a substantial number of common colds [ , ] . on the other hand, we now know of (at least) three zoonotic covs that recently have caused outbreaks in the human population: severe acute respiratory syndrome coronavirus (sars-cov) [ , ] in [ ] [ ] , middle east respiratory syndrome-coronavirus (mers-cov) [ , ] since (and probably earlier) and the current pandemic sars-cov- [ , ] . the latter agent emerged near wuhan (pr china) in the fall of and its animal source is currently under investigation [ ] [ ] [ ] . transmission to humans of sars-cov and mers-cov was attributed to civet cats [ ] and dromedary camels [ ] , respectively, although both species may have served merely as an intermediate host due to their close contact with humans. all three zoonotic covs belong to the genus betacoronavirus (beta-cov), which is abundantly represented among the covs that circulate in the many bat species on this planet [ , [ ] [ ] [ ] [ ] . the genetic diversity of bat covs and their phylogenetic relationships with the four known endemic hcovs (oc , hku , e and nl ; the latter two being alpha-covs) suggests that also these may have their evolutionary origins in bat hosts, for most of them probably centuries ago [ ] . the potential of multiple covs from different genera to cross species barriers had been predicted and documented previously [ , - , , ] , but regrettably was not taken seriously enough to invest more extensively in prophylactic and therapeutic solutions that could have contributed to rapidly containing an outbreak of the current magnitude. compared to other rna viruses, covs possess an unusually large positive-sense rna genome with a size ranging from to kilobases [ ] . the cov genome is single-stranded and its ′-proximal two-thirds encode for the large and partially overlapping replicase polyproteins pp a and pp ab ( - and - amino acids long, respectively), with the latter being a c-terminally extended version of the former that results from ribosomal frameshifting. the replicase polyproteins are processed into cleavage products (non-structural proteins, nsps) by two internal proteases, the papain-like protease (pl pro ) in nsp and the c-like or 'main' protease (m pro ) in nsp [ ] . specific transmembrane nsps (nsp , and ) then cooperate to transform intracellular membranes into a viral replication organelle (ro) [ ] that serves to organize and execute cov rna synthesis, which entails genome replication and the synthesis of an extensive nested set of subgenomic mrnas. the latter are used to express the genes present in the ′-proximal third of the genome, which encode the four common cov structural proteins [spike (s), envelope (e), membrane (m) and nucleocapsid (n) protein] and the 'so-called' accessory protein genes, most of which are thought to be involved in the modulation of host responses to cov infection [ ] . the cov proteome includes a variety of potential targets for drug repurposing or de novo development of specific inhibitors of, e.g. viral entry (s protein) or rna synthesis [ ] . the latter process depends on a set of enzymatic activities [ ] including an rna-dependent rna polymerase (rdrp; in nsp ), rna helicase (in nsp ), two methyltransferases involved in mrna capping (a guanine-n -methyltransferase in nsp and a nucleoside- ′-o-methyltransferase in nsp ) and a unique exoribonuclease (exon, in nsp ) that promotes the fidelity of the replication of the large cov genome [ ] . other potential drug targets are the transmembrane proteins that direct the formation of the viral ro, several less well characterized enzymatic activities and a set of smaller nsps (nsp - ) that mainly appear to serve as cofactors/modulators of other nsps. the newly emerged sars-cov- was rapidly identified as a cov that is relatively closely related to the sars-cov [ , , ] . the two genome sequences are about ~ % identical and the organization of orfs is essentially the same. the overall level of amino acid sequence identity of viral proteins ranges from about % in the least conserved parts of the s protein to about % in the most conserved replicative enzyme domains, prompting the coronavirus study group of the international committee on the taxonomy of viruses to classify the new agent within the species severe acute respiratory syndrome-related coronavirus, which also includes the sars-cov [ ] . the close phylogenetic relationship also implies that much of our knowledge of sars-cov molecular biology, accumulated over the past years, can probably be translated to sars-cov- . many reports posted over the past months have described such similarities, including the common affinity of the two viruses for the angiotensinconverting enzyme (ace ) receptor [ , ] . this receptor is abundantly expressed in vero cells (african green monkey kidney cells). since , vero cells have been used extensively for sars-cov research in cell-culture-based infection models by many laboratories, including our own. we set out to establish the basic features of sars-cov- replication in vero cells and compare it to the frankfurt- sars-cov isolate from [ , ] . when requesting virus isolates (february ), and in spite of the rapidly emerging public health crisis, we were confronted -not for the first time -with administrative hurdles and discussions regarding the alleged 'ownership' of virus isolates cultured from (anonymous) clinical samples. from a biological and evolutionary point of view, this would seem a strangely anthropocentric consideration, but it ultimately forced us to reach out across the globe to australian colleagues in melbourne. after checking our credentials and completing a basic material transfer agreement, they provided us (within week) with their first sars-cov- isolate (originally named -ncov/victoria/ / and subsequently renamed betacov/australia/vic / [ ] , which will be used throughout this study. until now, this isolate has been provided to other laboratories worldwide to promote the rapid characterization of sars-cov- , in this critical time of lockdowns and other preventive measures to avoid a collapse of public health systems. in this report, we describe a comparative study of the basic replication features of sars-cov and sars-cov- in vero e cells, including growth kinetics, virus titres, plaque phenotype and an analysis of intracellular viral rna and protein synthesis. additionally, we analysed infected cells by light and electron microscopy, and demonstrated cross-reactivity of available sars-cov-specific antisera (recognizing ten different viral proteins) with their sars-cov- counterparts. finally, we established the conditions for a mediumthroughput assay to evaluate basic antiviral activity and assessed the impact of some known cov inhibitors on sars-cov- replication. in addition to many anticipated similarities, our results also established some remarkable differences between the two viruses that warrant further investigation. one of them is the rapid evolution -during virus passaging in vero cells -of a specific region of the sars-cov- s protein that contains the so-called furin-like cleavage site. vero e cells and huh cells were grown as described previously [ ] . sars-cov- isolate australia/vic / (genbank id: mt . [ ] ) was derived from a positively testing nasopharyngeal swab in melbourne, australia, and was propagated twice in vero/hslam cells, before being shared with other laboratories. in leiden, the virus was passaged two more times at low m.o.i. in vero e cells to obtain a working stock (p stock) that was used in all experiments. sars-cov isolate frankfurt [ ] was used to compare growth kinetics and other features with sars-cov- . infection of vero e cells was carried out in pbs containing µg ml − deaedextran and % fcs (bodinco). the inoculum was added to the cells for h at °c, after which cells were washed twice with pbs and maintained in eagle's minimal essential medium (emem; lonza) with % fcs, mm l-glutamine (paa) and antibiotics (sigma). viral titres were determined by plaque assay in vero e cells as described previously [ ] . for plaque picking, plaque assays were performed using our p stock, while using an overlay containing % of agarose instead of avicel (rc- ; fmc biopolymer). following neutral red staining, small and large plaques were picked and used to inoculate a cm dish of vero e cells containing ml of emem- %fcs medium, yielding p virus. after h, µl of the culture supernatant was used to infect the next dish of cells (p ), a step that was repeated one more time to obtain p virus. all work with live sars-cov and sars-cov- was performed in biosafety laboratory level facilities at leiden university medical center, the netherlands. isolation of intracellular rna was performed by lysing infected cell monolayers with tripure isolation reagent (roche applied science) according to the manufacturer's instructions. after purification and ethanol precipitation, intracellular rna samples were loaded onto a . % agarose gel containing . m formaldehyde, which was run overnight at low voltage in mops buffer [ mm mops (sodium salt) (ph ), mm sodium acetate, mm edta]. dried agarose gels were used for direct detection of viral mrnas by hybridization with a p-labelled oligonucleotide probe ( ′-cacatggggatagcactac- ′) that is complementary to a fully conserved sequence located nucleotides upstream of the ' end of the genome as well as all subgenomic mrnas produced by sars-cov- and sars-cov. after hybridization, rna bands were visualized and quantified by phosphorimaging using a typhoon- variable mode scanner (ge healthcare) and imagequant tl software (ge healthcare). in order to verify the amount of rna loaded, a second hybridization was performed using a p-labelled oligonucleotide probe recognizing s ribosomal rna ( ′-gatc cgag ggcc tcac taaac- ′). protein lysates were obtained by lysing infected cell monolayers in ×laemmli sample buffer and were analysed by semi-dry western blotting onto hybond . µm polyvinylidene difluoride (pvdf) membrane (ge healthcare). membranes were incubated with rabbit antisera diluted in pbs with . % tween- containing % dry milk (campina). primary antibodies were detected with a horseradish peroxidase-conjugated swine anti-rabbit igg antibody (dako) and protein bands were visualized using clarity western blot substrate (biorad) and detected using an advanced q alliance imager (uvitec cambridge). sars-cov- genomic rna was isolated from cell-culture supernatants using tripure isolation reagent (roche applied science) and purified according to the manufacturer's instructions. the total amount of rna in samples was measured using a qubit fluorometer and rna high sensitivity kit (thermo fisher scientific). for next-generation sequencing (ngs) library preparation, rna ( - ng) was mixed with random oligonucleotide primers using the nebnext first strand synthesis module kit for illumina (neb) and incubated for min at °c. ngs of samples was performed by a commercial service provider (genomescan, leiden, the netherlands) while including appropriate quality controls after each step of the procedure. sequencing was performed using a novaseq sequencing system (illumina). subsequently, sequencing reads were screened for the presence of human (grch . ), mouse (grcm .p ), e. coli mg (embl u . ), phix (refseq nc_ . ) and common vector sequences (univec and chlsab . ). prior to alignment, reads were trimmed to remove adapter sequences and filtered for sequence quality. the remaining reads were mapped to the sars-cov- genbank reference sequence (nc_ . [ ] ). data analysis was performed using bowtie [ ] . raw ngs data sets for each virus sample analysed in this study are deposited in ncbi bioproject and available under the following link: http://www. ncbi. nlm. nih. gov/ bioproject/ . only sars-cov- -specific reads were included in these data files. to study evolution/adaptation of the s protein gene, we performed an in-depth analysis of reads covering the s /s region of the s protein gene. this was done for the p stock and for the four virus samples of the plaque-picking experiment shown in fig. a . first, all reads spanning nt to of the sars-cov- genome were selected. next, reads constituting less than % of the total number of selected reads were excluded from further analysis. the remaining number of reads were (p stock), (s p ), (s p ), (s p ) and (l p ). these reads were translated in the s protein orf and the resulting amino acid sequences were aligned, grouped on the basis of containing the same mutations/deletions in the s /s region and ranked by frequency of occurrence (fig. b) . the sars-cov-specific rabbit or mouse antisera/antibodies used in this study are listed in table . most antisera were described previously (see references in table ), with the exception of three rabbit antisera recognizing sars-cov nsps , and . these were raised using full-length (his) -tagged table . sars-cov-specific antisera used and their cross-reactivity with corresponding sars-cov- targets antigen type antibody type ifa signal* reference nsp (dgd ) transmembrane replicase protein, containing pl pro bacterial expression product rabbit polyclonal ++ [ ] nsp (fgq ) transmembrane replicase protein synthetic peptide rabbit polyclonal ++ [ ] nsp (due ) m pro bacterial expression product rabbit polyclonal + [ ] nsp (gbz ) transmembrane replicase protein synthetic peptide rabbit polyclonal − [ ] nsp (duk ) rna polymerase co-factor bacterial expression product rabbit polyclonal ++ [ ] nsp ( * ++, strongly positive; +, positive; -, negative. following a plaque assay of the p virus stock, small and large plaques were picked and these virus clones were passaged three times in vero e cells, while their plaque phenotype was monitored. in contrast to the large plaque viruses (example l ; bottom row), the plaque phenotype of the small plaque viruses (example s ; top row) rapidly evolved within these three passages. (b) evolution/adaptation of the s protein gene during vero e passaging. overview of ngs data obtained for the p stock, s p /p /p and s p in the s /s region of the sars-cov- s protein gene that encodes the so-called furin-like cleavage site. the analysis was based on ngs reads spanning nt to of the sars-cov genome (see methods for details) and their translation in the s protein orf. deletions are indicated with Δ followed by the affected amino acid residues. bacterial expression products (nsp and nsp ) or a synthetic peptide (nsp , aa - of sars-cov pp a), which were used to immunize new zealand white rabbits as described previously [ , ] . cross-reactivity of antisera to sars-cov- targets was evaluated microscopically by immunofluorescence assay (ifa) and for some antisera (nsp and n protein) also by western blot analysis. double-stranded rna was detected using mouse monoclonal antibody j from scicons [ ] . cells were grown on glass coverslips and infected as described above [ ] . at sars-cov- isolate betacov/australia/vic / was received as a stock derived from two consecutive passages in vero/hslam cells [ ] . the virus was then propagated two more times at low m.o.i. in vero e cells, in which it caused a severe cytopathic effect (cpe). we also attempted propagation in huh cells, using the same amount of virus or a tenfold larger inoculum, but did not observe any cytopathology after h (data not shown [ ] and other field isolates [ ] , isolate betacov/australia/vic / exhibits > . % sequence identity. in addition to synonymous mutations in the nsp -coding sequence (u to c) and s protein gene (u to g), orf a contains a single non-synonymous mutation (g to u). strikingly, the ′ utr contains a nt deletion (nt - ; cgaucgagug) located nt upstream of the genomic ′ end, which is not present in other sars-cov- isolates described thus far (> sars-cov sequences present in genbank on april ). in about % of the p ngs reads covering this position, we noticed a g to a mutation encoding an arg to gln substitution near the so-called s /s cleavage site of the viral s protein (see discussion), with the other % of the reads being wild-type sequence. as this ratio approximated the observed relative proportions between large and small plaques, we performed a plaque assay on the p virus stock (fig. a, leftmost well) and picked multiple plaques of each size, which were passaged three times in vero e cells while monitoring their plaque phenotype. interestingly, for several of the small-plaque virus clones (like s ; fig. a ) we observed rapid conversion to a mixed or large-plaque phenotype during these three passages, while large-plaque virus clones (like l ) stably retained their plaque phenotype (fig. a) . ngs analysis of the genome of a large-plaque p virus (l p ) revealed that > % of the reads in the s /s cleavage site region contained the g to a mutation described above. no other mutations were detected in the genome, thus clearly linking the arg to gln substitution in the s protein to the large-plaque phenotype observed for the l p virus. next, we also analysed the genomes of the p , p and p viruses derived from a small-plaque (s ) that was picked. this virus clone retained its small-plaque phenotype during the first passage ( fig. a; s p ), but began to yield an increasing proportion of large(r) plaques during subsequent passages. sequencing of s p (fig. b) revealed a variety of lowfrequency reads with mutations near the s /s cleavage site motif (aa - ; prrar↓sv), with g to a (specifying the arg to gln substitution) again being the dominant one (in ~ . % of the reads covering nt to of the genome). at lower frequencies single-nucleotide changes specifying arg to trp and arg to leu substitutions were also detected. furthermore, a aa deletion (residues - ) that erases the s /s cleavage site region was discovered, as well as a aa deletion (residues - ) immediately preceding that region. the amount of large plaques increased substantially upon the next passage, with ngs revealing the prominent emergence of the mutants containing the aa deletion or the arg to gln point mutation (~ and~ % of the reads, respectively), and yet other minor variants with mutations in the prrar↓sv sequence being discovered. taken together these data clearly link the large-plaque phenotype of sars-cov- to the acquisition of mutations in this particular region of the s protein, which apparently provides a strong selective advantage during passaging in vero e cells. to our knowledge, a detailed comparison of sars-cov- and sars-cov replication kinetics in cell culture has not been reported so far. therefore, we infected vero e cells with the sars-cov- /p virus stock at high m.o.i. to analyse viral rna synthesis and the release of infectious viral progeny (fig. a) . this experiment was performed using four replicates per time point and for comparison we included the sars-cov frankfurt- isolate [ ] , which has been used in our laboratory since . during the early stages of infection (until h p.i.), the growth curves of the two viruses were similar, but subsequently cells infected with sars-cov clearly produced more infectious progeny (about -fold more) than sars-cov- -infected cells, with both viruses reaching their plateau by about h p.i. as shown in fig. b , despite its transition to a mainly large-plaque phenotype, the largest sars-cov- /p plaques were still substantially smaller than those obtained with sars-cov frankfurt- . in parallel, we analysed the kinetics of viral rna synthesis by isolating intracellular viral rna, subjecting it to agarose gel electrophoresis and visualizing the various viral mrna species by in-gel hybridization with a p-labelled oligonucleotide probe recognizing a fully conserved nt sequence located nt upstream of the ′ end of both viral genomes (fig. a) . this revealed the anticipated presence of the genomic rna and eight subgenomic mrnas, together forming the well-known ′-and ′-coterminal nested set of transcripts required for full cov genome expression. in general, for both viruses, the accumulation of viral rnas followed the growth curves depicted in fig. a . the relative abundance of the individual rnas was determined using the , and h p.i. samples (averages presented in fig. b ) and found to be largely similar, with the exception of sars-cov- mrnas and , which accumulated to about four and twofold higher levels, respectively. strikingly, in spite of the ultimately lower yield of infectious viral progeny, sars-cov- rna synthesis was detected earlier and reached an overall level exceeding that of sars-cov. overall, we conclude that in vero e cells, sars-cov- produces levels of intracellular rna that are at least comparable to those of sars-cov, although this does not translate into the release of equal amounts of infectious viral progeny (fig. a) . to be able to follow virus replication in sars-cov- -infected cells more closely, we explored cross-reactivity of a variety of antisera previously raised against sars-cov targets, in particular a variety of nsps. in an earlier study, many of those were found to cross-react also with the corresponding mers-cov targets [ ] , despite the relatively large evolutionary distance between mers-cov and sars-cov. based on the much closer relationship with sars-cov- , similar or better cross-reactivity of these sars-cov reagents was expected, which was explored using immunofluorescence microscopy. indeed, most antisera recognizing sars-cov nsps that were tested (nsp , nsp , nsp , nsp , nsp , nsp , nsp ) strongly cross-reacted with the corresponding sars-cov- target (fig. , table ), the exception being a polyclonal nsp rabbit antiserum. likewise, both a polyclonal rabbit antiserum and mouse monoclonal antibody recognizing the n protein cross-reacted strongly (fig. b , table ). the same was true for a rabbit antiserum raised against a c-terminal peptide of the sars-cov m protein (fig. e) . labelling patterns were essentially identical to those previously documented for sars-cov [ , ] , with nsps accumulating in the perinuclear region of infected cells, where the elaborate membrane structures of the viral ros are formed (fig. a, c and d) . punctate structures in the same area of the cell were labelled using an antibody recognizing double-stranded rna (dsrna), which presumably recognizes replicative intermediates of viral rna synthesis [ , ] . the n protein signal was diffusely cytosolic (fig. b) , whereas the m protein labelling predominantly showed the expected localization to the golgi complex (fig. e) , where the protein is known to accumulate [ ] . we next used electron microscopy to investigate the ultrastructural changes that sars-cov- induces in infected cells, and focused on the membranous replication organelles (ros) that support viral rna synthesis and on the assembly and release of new virions (fig. ) . compared to mock-infected control cells (fig. a-b) , various distinct membrane alterations were observed in cells infected with either sars-cov or sars-cov- ( fig. c-j) . at h p.i., larger regions with membrane alterations were found particularly in cells infected with sars-cov- (data not shown), which may align with the somewhat faster onset of intracellular rna synthesis in sars-cov -infected vero e cells (fig. a) . from h p.i. onwards, sars-cov-and sars-cov- -infected cells appeared more similar (fig. c-j) . double-membrane vesicles (dmvs) were the most prominent membrane alteration up to this stage (fig. d -e, h-i). in addition, convoluted membranes [ ] were readily detected in sars-cov-infected cells, while zippered er [ , , ] appeared to be the predominant structure in sars-cov- -infected cells (fig. e , i, white arrowheads). as previously described for sars-cov [ ] , sars-cov- -induced dmvs also appeared to fuse through their outer membrane, giving rise to vesicle packets that increased in numbers as infection progressed (fig. f , k, white asterisks). virus budding near the golgi apparatus, presumably into smooth membranes of the er-golgi intermediate compartment (ergic) [ , , ] , was frequently observed at h p.i. (fig. k-l, o-p) . this step is followed by transport to the plasma membrane and release of virus particles into extracellular space. by h p.i., released progeny virions were abundantly detected around all infected cells (fig. m -n, q-r). interestingly, whereas spikes were clearly present on sars-cov progeny virions, a relatively large proportion of sars-cov- particles seemed to carry few or no visible spike projections on their surface, perhaps suggesting a relatively inefficient incorporation of spike proteins into sars-cov- virions. this could potentially reduce the yield of infectious particles and may contribute to the lower progeny titres obtained for this virus (fig. a) . in order to establish and validate a cpe-based assay to identify potential inhibitors of sars-cov- replication, we selected four previously identified inhibitors of cov replication: remdesivir [ , ] , chloroquine [ , ] , alisporivir [ , ] and pegylated interferon alpha (peg-ifn-α) [ , ] [ , ] respectively; fig. a ) than previously reported by others, but this may be explained by technical differences like a longer assay incubation time ( h instead of h) and the use of a different read-out (cell viability instead of qrt-pcr or viral load). based on the obtained half maximal cytotoxic concentration (cc ) values of > µm, a selectivity index > . was calculated. chloroquine potently blocked virus infection at low-micromolar concentrations, with an ec value of . ± . µm for both viruses (cc > µm, si> . ; fig. b ). alisporivir, a known inhibitor of different groups of rna viruses, was previously found to effectively reduce the production of cov progeny. in this study, we measured ec values of . ± . and . ± . µm for sars-cov- and sars-cov, respectively ( fig. c ; cc > µm, si> ). treatment with peg-ifn-α completely inhibited replication of sars-cov- , even at the lowest dose of . ng ml − (fig. d ). in line with previous results [ , ] , sars-cov was much less sensitive to peg-ifn-α treatment, yielding only partial inhibition at all concentrations tested (from . to ng ml − ). overall, we conclude that vero e cells provide a suitable basis to perform antiviral compound screening and select the most promising hits for in-depth mechanistic studies and further development. in this report, we describe a comparative analysis of the replication features of sars-cov- and sars-cov in vero e cells, one of the most commonly used cell lines for studying these two viruses. in contrast to the stable phenotype exhibited by sars-cov during our years of working with this virus in these cells, sars-cov- began to exhibit remarkable phenotypic variation in plaque assays within a few passages after its isolation from clinical samples (fig. a) . in addition to the betacov/australia/vic / isolate used in this study, similar observations were made for a variety of other clinical isolates (data not shown). to establish the genetic basis for the observed plaque size heterogeneity, small and large plaques were picked and the resulting virus clones were passaged repeatedly and analysed using ngs. the consensus sequences obtained for s p and l p , which differed by a single nucleotide substitution in the s protein gene, clearly established that a single s protein mutation (arg to gln) was responsible for the observed plaque size difference. this mutation is localized near the so-called furin-like s /s cleavage site (fig. b) [ ] in the s protein [ ] . this sequence constitutes a (potential) processing site that is present in a subset of covs (including sars-cov- and mers-cov) but is lacking in others, like sars-cov and certain bat covs [ , ] . this polybasic motif (prrar↓sv, in sars-cov- ) can be recognized by intracellular furin-like proteases during viral egress and its cleavage is thought to prime the s protein for fusion and entry [ ] , which also requires a second cleavage event to occur at the downstream s ' cleavage site [ ] . in general, the presence of the furin-like cleavage site does not appear to be critical for successful cov infection. using pseudotyped virions carrying mutant s proteins of sars-cov [ ] or sars-cov- [ ] , it was shown that its presence minimally impacts s protein functionality. in the sars-cov s protein, an adjacent sequence that is conserved across covs can be cleaved by other host proteases like cathepsin l or tmprss [ ] [ ] [ ] , thus providing an alternative pathway to trigger viral entry. possibly, this pathway is also employed by our vero e -cell adapted sars-cov- mutants that have lost the furin-like cleavage site, like clone l p and multiple variants encountered in s p (fig. a) . these variants contain either single point mutations or deletions of to aa (fig. b) , resembling variants recently reported by other laboratories [ , , ] . interestingly similar changes were also observed in some clinical sars-cov- isolates that had not been passaged in cell culture [ ] . it is currently being investigated why mutations that inactivate the furin-like cleavage site provide such a major selective advantage during sars-cov- passaging in vero e cells and how this translates into the striking large-plaque phenotype documented in this paper. an additional remarkable feature confirmed by our re-sequencing of the betacov/australia/vic / isolate of sars-cov- is the presence of a nt deletion in the ′ utr of the genome [ ] . screening of other available sars-cov- genome sequences indicated that the presence of this deletion apparently is unique for this particular isolate, and likely represents an additional adaptation acquired during cell-culture passaging. this deletion maps to a previously described 'hypervariable region' in the otherwise conserved ′ utr, and in particular to the so-called s m motif [ ] that is conserved among covs and also found in several other virus groups [ , ] . the s m element has been implicated in the binding of host factors to viral rnas, but its exact function has remained enigmatic thus far. strikingly, for the mouse hepatitis coronavirus the entire hypervariable region (including s m) was found to be dispensable for replication in cell culture, but highly relevant for viral pathogenesis in mice [ ] . although the impact of this deletion for sars-cov- remains to be studied in more detail, these previous data suggest that this mutation need not have a major impact on sars-cov- replication in vero e cells. this notion is also supported by the fact that the results of our antiviral screening assays (fig. ) correlate well with similar studies performed with other sars-cov- isolates [ , , ] . clearly, this could be different for in vivo studies, for which it would probably be better to rely on sars-cov- isolates not carrying this deletion in their ′ utr. vero e cells are commonly used to isolate, propagate and study sars-cov-like viruses as they support viral replication to high titres [ ] [ ] [ ] [ ] [ ] . this may be due to a high expression level of the ace- receptor [ ] that is used by both sars-cov- and sars-cov [ ] and/or the fact that they lack the ability to produce interferon [ , ] . it will be interesting to evaluate whether there is a similarly strong selection pressure to adapt the s /s region of the s protein when sars-cov- is passaged in other cell types. such studies are currently in progress in our laboratory and already established that huh cells may be a poor choice, despite the fact that they were used for virus propagation [ , ] and antiviral screening in other studies [ , ] . immunolabelling of infected huh cells (data not shown) revealed non-productive infection of only a small fraction of the cells and a general lack of cytopathology. while other cell lines are being evaluated, the monitoring of the plaque phenotype (plaque size and homogeneity) as illustrated above may provide a quick and convenient method to assess the composition of sars-cov- stocks propagated in vero e cells, at least where it concerns the evolution of the s /s region of the s protein. given the ongoing sars-cov- pandemic, the detailed characterization of its replication cycle is an important step in understanding the molecular biology of the virus and defining potential targets for inhibitors of replication. the cross-reacting antisera described in this study (table ) will be a useful tool during such studies. in general, the subcellular localization of viral nsps and structural proteins (fig. ) and the ultrastructural changes associated with ro formation (fig. ) were very similar for the two viruses. we also observed comparable replication kinetics for sars-cov- and sars-cov in vero e cells, although clearly lower final infectivity titres were measured for sars-cov- (~ -fold lower; fig. ). nevertheless, rna synthesis could be detected somewhat earlier for sars-cov- and the overall amount of viral rna produced exceeded that produced by sars-cov (fig. ) . this may be indicative of certain assembly or maturation problems or of virus-host interactions that are different in the case of sars-cov- . these possibilities merit further investigation, in particular since our preliminary em studies suggested intriguing differences with sars-cov regarding the abundance of spikes on the surface of freshly released sars-cov- particles (fig. n, r) . our analysis of sars-cov- subgenomic mrna synthesis revealed an increased relative abundance of mrnas and (~four and ~twofold, respectively) in comparison to sars-cov. mechanistically, these differences do not appear to be caused by extended base-pairing possibilities of the transcription regulatory sequences that direct the synthesis of these two mrnas [ ] . as in sars-cov, mrna of sars-cov- encodes for two proteins, the orf a and orf b proteins, with the latter presumably being expressed following leaky ribosomal scanning [ ] . upon ectopic expression, the orf a protein has been reported to induce apoptosis via a caspasedependent pathway [ ] and/or to be involved in cell-cycle arrest [ ] . the orf b product is a poorly studied integral membrane protein that has (also) been detected in virions [ ] . when orf a/b or orf a were deleted from the sars-cov genome, there was a minimal impact on the kinetics of virus replication in vitro in different cell lines, including vero cells, and in vivo using mice. in another study, however, partial deletion of sars-cov orf b was reported to provide a replicative advantage in caco- and huh cells, but not in vero cells [ ] . the sars-cov orf protein is membrane-associated and able to induce endoplasmic reticulum stress [ , ] , although it has not been characterized in great detail in the context of viral infection. soon after the emergence of sars-cov in , a conspicuous nt (out-offrame) deletion in orf was noticed in late(r) human isolates, but not in early human isolates and sars-like viruses obtained from animal sources [ ] [ ] [ ] . consequently, loss of orf function was postulated to reflect an adaptation to the human host. the re-engineering of an intact orf , using a reverse genetics system for the sars-cov frankfurt- isolate, yielded a virus with strikingly enhanced (up to -fold) replication properties in multiple systems [ ] . clearly, it remains to be established whether the increased synthesis of mrnas and is a general feature of sars-cov- isolates, and this indeed also translates into higher expression levels of the accessory proteins encoded by orfs a, b and . if confirmed, these differences definitely warrant an in-depth follow-up analysis as cov accessory proteins in general have been shown to be important determinants of virulence. they may thus be relevant for our understanding of the wide spectrum of respiratory disease symptoms observed in covid- patients [ ] . based on the close ancestral relationship between sars-cov- and sars-cov [ ] , one might expect that the patterns and modes of interaction with host antiviral defence mechanisms would be similar. however, our experiments with type-i interferon treatment of vero e cells (fig. ) revealed a clear difference, with sars-cov- being considerably more sensitive than sars-cov, as also observed by other laboratories [ ] . essentially, sars-cov- replication could be inhibited by similarly low concentrations of peg-ifn-alpha- a that inhibit mers-cov replication in cell culture [ ] . taken together, our data suggest that sars-cov- is less able to counteract a primed type-i ifn response than sars-cov [ , ] . previously identified inhibitors of cov replication were used to further validate our cell-based assay for sars-cov- inhibitor screening. these compounds inhibited replication at similar low-micromolar concentrations and in a similar dose-dependent manner as observed for sars-cov (fig. ). remdesivir is a prodrug of an adenosine analogue developed by gilead sciences. it was demonstrated to target the cov rna polymerase and act as a chain terminator [ ] [ ] [ ] . the clinical efficacy of remdesivir is still being evaluated and, after some first encouraging results [ ] , worldwide compassionate use trials are now being conducted. likewise, hydroxychloroquine and chloroquine have been labelled as potential 'game changers' and are being evaluated for treatment of severe covid- patients [ ] . both compounds have been used to treat malaria and amebiasis [ ] , until drug-resistant plasmodium strains emerged [ ] . these compounds can be incorporated into endosomes and lysosomes, raising the ph inside these intracellular compartments, which in turn may lead to defects in protein degradation and intracellular trafficking [ , ] . an alternative hypothesis to explain their anti-sars-cov activity is based on their impact on glycosylation of the ace receptor that is used by sars-cov [ ] . finally, as expected, the non-immunosuppressive cyclosporin a analogue alisporivir inhibited sars-cov- replication, as demonstrated previously for sars-cov and mers-cov [ ] . although the exact mode of action of this inhibitor is unclear, it is thought to modulate cov interactions with members of the cyclophilin family [ ] . unfortunately, all of these in vitro antiviral activities should probably be classified as modest, emphasizing the urgency of large-scale drug repurposing and discovery programmes that target sars-cov- and coronaviruses at large. the authors received no specific grant from any funding agency. coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- bat origin of a new human coronavirus: there and back again coronavirus infection in acute lower respiratory tract disease of infants identification of new human coronaviruses a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia genomic characterization of a 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infection in a mouse model pegylated interferon-alpha protects type pneumocytes against sars coronavirus infection in macaques assay guidance manual the spike glycoprotein of the new coronavirus -ncov contains a furin-like cleavage site absent in cov of the same clade the proteolytic regulation of virus cell entry by furin and other proprotein convertases a pneumonia outbreak associated with a new coronavirus of probable bat origin host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein furin cleavage of the sars coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry structure, function, and antigenicity of the sars-cov- spike glycoprotein cathepsin l functionally cleaves the severe acute respiratory syndrome coronavirus class i fusion protein upstream of rather than adjacent to the fusion peptide coronavirus cell entry occurs through the endo-/ lysosomal pathway in a proteolysis-dependent manner sars coronavirus, but not human coronavirus nl , utilizes cathepsin l to infect ace -expressing cells identification of a common deletion in the spike protein of sars-cov- characterisation of the transcriptome and proteome of sars-cov- using direct rna sequencing and tandem mass spectrometry reveals evidence for a cell passage induced in-frame deletion in the spike glycoprotein that removes the furin-like cleavage site a hypervariable region within the ' cis-acting element of the murine coronavirus genome is nonessential for rna synthesis but affects pathogenesis a conserved rna pseudoknot in a putative molecular switch domain of the '-untranslated region of coronaviruses is only marginally stable rna genome conservation and secondary structure in sars-cov- and sars-related viruses remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov- replication in vitro sars-cov- sensitive to type i interferon pretreatment isolation, sequence, infectivity and replication kinetics of sars-cov- enhanced isolation of sars-cov- by tmprss -expressing cells apical entry and release of severe acute respiratory syndromeassociated coronavirus in polarized calu- lung epithelial cells exogenous ace expression allows refractory cell lines to support severe acute respiratory syndrome coronavirus replication sars-associated coronavirus replication in cell lines discovery of novel human and animal cells infected by the severe acute respiratory syndrome coronavirus by replicationspecific multiplex reverse transcription-pcr studies on the mechanism of the priming effect of interferon on interferon production by cell cultures exposed to poly(ri)-poly(rc) regulation of the interferon system: evidence that vero cells have a genetic defect in interferon production a novel coronavirus from patients with pneumonia in china the fdaapproved gold drug auranofin inhibits novel coronavirus (sars-cov- ) replication and attenuates inflammation in human cells overexpression of a, a protein specifically encoded by the severe acute respiratory syndrome coronavirus, induces apoptosis via a caspase-dependent pathway sars coronavirus a protein blocks cell cycle progression at g /g phase via the cyclin d /prb pathway the orf b protein of severe acute respiratory syndrome coronavirus (sars-cov) is expressed in virus-infected cells and incorporated into sars-cov particles the sars-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors the ab protein of sars-cov is a luminal er membrane-associated protein and induces the activation of atf sars-coronavirus open reading frame- b triggers intracellular stress pathways and activates nlrp inflammasomes molecular evolution of the sars coronavirus during the course of the sars epidemic in china isolation and characterization of viruses related to the sars coronavirus from animals in southern china severe acute respiratory syndrome (sars) coronavirus orf protein is acquired from sars-related coronavirus from greater horseshoe bats through recombination attenuation of replication by a nucleotide deletion in sars-coronavirus acquired during the early stages of human-to-human transmission understanding sars-cov- -mediated inflammatory responses: from mechanisms to potential therapeutic tools genomic characterization of the novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan inhibition of novel β coronavirus replication by a combination of interferon-α b and ribavirin the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus structural basis for inhibition of the rna-dependent rna polymerase from sars-cov- by remdesivir remdesivir and sars-cov- : structural requirements at both nsp rdrp and nsp exonuclease active-sites first case of novel coronavirus in the united states a rush to judgment? rapid reporting and dissemination of results and its consequences regarding the use of hydroxychloroquine for covid- in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) chloroquine-resistant malaria chloroquine analogues in drug discovery: new directions of uses, mechanisms of actions and toxic manifestations from malaria to multifarious diseases cyclophilins and cyclophilin inhibitors in nidovirus replication sars-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro coronaviruses use discontinuous extension for synthesis of subgenome-length negative strands genome organization of the sars-cov we thank various genomescan staff members for the pleasant and swift collaboration that facilitated the ngs and data analysis of the first sars-cov- samples. we are grateful to all members of the sections research and clinical microbiology of the lumc department of medical microbiology for their collaborative support and dedication during the current pandemic situation. in particular, we thank linda boomaars, peter bredenbeek, ien dobbelaar, martijn van hemert, sebenzile myeni, tessa nelemans, esther quakkelaar, ali tas, sjaak van voorden and gijsbert van willigen for their technical or administrative support, constructive discussions and/or scientific input. the authors declare that there are no conflicts of interest. five reasons to publish your next article with a microbiology society journal . the microbiology society is a not-for-profit organization. . we offer fast and rigorous peer review -average time to first decision is - weeks. . our journals have a global readership with subscriptions held in research institutions around the world. . % of our authors rate our submission process as 'excellent' or 'very good'. . your article will be published on an interactive journal platform with advanced metrics.find out more and submit your article at microbiologyresearch.org. key: cord- - cylm o authors: yamamoto, norio; yang, rongge; yoshinaka, yoshiyuki; amari, shinji; nakano, tatsuya; cinatl, jindrich; rabenau, holger; doerr, hans wilhelm; hunsmann, gerhard; otaka, akira; tamamura, hirokazu; fujii, nobutaka; yamamoto, naoki title: hiv protease inhibitor nelfinavir inhibits replication of sars-associated coronavirus date: - - journal: biochem biophys res commun doi: . /j.bbrc. . . sha: doc_id: cord_uid: cylm o a novel coronavirus has been identified as an etiological agent of severe acute respiratory syndrome (sars). to rapidly identify anti-sars drugs available for clinical use, we screened a set of compounds that included antiviral drugs already in wide use. here we report that the hiv- protease inhibitor, nelfinavir, strongly inhibited replication of the sars coronavirus (sars-cov). nelfinavir inhibited the cytopathic effect induced by sars-cov infection. expression of viral antigens was much lower in infected cells treated with nelfinavir than in untreated infected cells. quantitative rt-pcr analysis showed that nelfinavir could decrease the production of virions from vero cells. experiments with various timings of drug addition revealed that nelfinavir exerted its effect not at the entry step, but at the post-entry step of sars-cov infection. our results suggest that nelfinavir should be examined clinically for the treatment of sars and has potential as a good lead compound for designing anti-sars drugs. severe acute respiratory syndrome (sars) is an emerging disease that was first reported in guangdong province, people's republic of china, in november, . since then, sars has spread to countries and has resulted in more than deaths from respiratory distress syndrome [ ] [ ] [ ] . an overall estimate of case fatality reached - % as reported by who [ ] and the mortality rate in people older than years could be as high as - % [ ] . several groups, including the authors, isolated a novel coronavirus from sars patients [ , , ] . it has been shown that sars-cov satisfies koch's postulates for causation-its consistent isolation from patients suffering from sars, isolation of the virus and reproduction of disease in non-human primates after inoculation, and the presence of a specific antibody response against the virus in both sars patients and artificially infected primates [ ] . now its etiological role in sars is widely accepted. the outbreak of sars in several countries has led to the search for active antiviral compounds and vaccines for this disease [ ] . although the results of many clinical experiments have been reported, no consensus on treatment has been reached to date. therapeutic protocols with steroids and ribavirin have been widely used empirically from the outset of the epidemic [ , ] . the use of steroids for sars seemed beneficial, whenever they are appropriately applied. however, the optimal timing, dosage, and duration of treatment have not yet been determined. on the other hand, the administration of ribavirin did not apparently reduce either the rate of intratracheal intubation or that of mortality [ ] . moreover, significant toxicity, such as hemolytic anemia, has been attributed to ribavirin [ ] . a few preliminary trials and in vitro data suggested the possibility of treating sars with interferon [ ] [ ] [ ] . other agents including glycyrrhizin and convalescent plasma require further studies [ ] . as is well established in the case of hiv- infection, the combination of antiviral drugs will make it possible to establish a better protocol for the treatment of sars. to identify anti-sars drugs available for clinical use as rapidly as possible, we screened a set of compounds including antiviral drugs already in human clinical use. we found that nelfinavir, a widely used hiv- protease inhibitor, could inhibit sars-cov replication efficiently. our results suggest that nelfinavir should be examined clinically for the treatment of sars. cell culture and virus. vero e cells were maintained in dulbecco's modified eagle's medium supplemented with % fbs and glutaminepenicillin-streptomycin solution in % co in humidified air at °c. the ffm- strain of sars-cov was isolated from a sars patient admitted to the clinical centre of frankfurt university. this strain was used in all experiments to assess the antiviral activity of the drugs. compounds for screening. a set of compounds for screening consisted of drugs as follows: nelfinavir, saquinavir, kni- , tya , tyb , ritonavir, lopinavir, indinavir, f-benzoyl-tn , f-benzoyl-te , tn , t , tc , fc , t , sdf- , vmip-ii, tak- , sc , n , t- , glycyrrhizin, glycyrrhetic acid, and cardran sulfate. cytopathic effect assay. sars-cov was inoculated into a monolayer of vero e cells in -well plates at a multiplicity of infection (moi) of . . the plates were incubated at °c in % co for days and cpe in each well was observed. immunofluorescence assay. the vero e cells in -well plates were infected with sars-cov at the moi of . . the infected cells were fixed with methanol h after infection and incubated at room temperature for h with diluted serum sample from a sars patient. after washing with pbs, the cells were incubated with anti-human-igg antibody conjugated with fitc for min at room temperature. the cells were washed with pbs, mounted in buffered glycerol, cover-slipped, and viewed with a fluorescence microscope. rna extraction and real-time rt-pcr assay. sars-cov rna in the culture supernatant was purified with isogen (nippongene) according to the manufacturer's protocol. for quantification of sars-cov orf- rna, we performed real-time rt-pcr with the primers and the probe as follows: orf -f, agctacgagcaccagacacc; orf -r, actttgggcattccccttt; orf -probe, tcgaaa ttaagagtgccaagaaatttgacacttt. the fluorescence intensity generated from the probe was detected by the abi- sequence detector system (applied biosystems). mtt assay. vero e cells in -well plates were infected with sars-cov at the moi of . . after h of culture, cells were incubated for h in the presence of . mg/ml of -( , -dimethylthiazol- -yl)- , -diphenyl tetrazolium bromide (mtt). formazan crystals were dissolved with ll of . n hcl-isopropyl alcohol (acid isopropanol) and absorbance at nm was measured with a reference wavelength of nm. time-of-addition experiments. drugs, including nelfinavir, were added to cultures of vero e cells at the time of infection or h after infection. samples were processed for a quantitative rt-pcr assay and an immunofluorescence assay h after infection. the cytopathic effect of infected cells was analyzed h after infection. entry inhibition assay. vero e cells were pretreated with each drug for h, and sars-cov was inoculated at the moi of . . cells and viruses were incubated for h and washed with pbs three times. subsequently, infected cells were lysed with isogen (nippongene) and rna was purified according to the manufacturer's protocol. extracted rna samples were subjected to real-time rt-pcr analysis for quantification of sars-cov rna as described above. as a loading control for normalization, s ribosomal rna was quantified with the primers and the probe as follows: s-f, gtaacccgttgaaccccatt; s-r, ccatccaatcggtagtagcg; and s-probe, tgcgtt gattaagtccctgccctttgta. we screened our chemical library and found that nelfinavir could inhibit sars-cov replication in vero e cells. nelfinavir clearly inhibited the cytopathic effect (cpe) induced by infection with sars-cov (fig. a) . we also examined the replication of sars-cov by immunofluorescence assay (ifa) with a serum sample from a patient with sars. expression of viral antigens was much lower in infected cells treated with nelfinavir than in untreated infected cells (fig. b) . furthermore, we assessed the effect of nelfinavir on the production of virions. nelfinavir significantly blocked the production of virions as revealed by quantitative rt-pcr (fig. ) . by the use of mtt assay, we determined the concentration of the compound that reduced cell viability to % (cc ), the concentration of the compound required for inhibition of cpe to % of the control value (ec ), and the selectivity index (si). nelfinavir inhibited sars-cov replication at non-toxic doses with an approximate si of , while the other inhibitors against hiv- protease (ritonavir, lopinavir, saquinavir, indinavir, tya , tyb , and kni- ) did not affect the replication of sars-cov (table ). these results revealed that nelfinavir is active in inhibiting sars-cov replication. nelfinavir inhibited sars-cov replication at the postentry, but not the entry step to disclose the step at which nelfinavir affects the virus life cycle, we performed time-of-addition experiments on the replication of sars-cov. nelfinavir significantly inhibited sars-cov replication when used before infection (figs. a and b and ) . when this drug was added at the time of infection or h after infection, it was still able to block the cpe induced by sars-cov infection (fig. a) . addition of nelfinavir at various timings inhibited the expression of viral antigens in vero cells as shown by ifa (fig. b) . nelfinavir blocked the production of virions when used to treat the cells at the time of infection or h after infection (fig. ) . the other protease inhibitors including ritonavir had no effect on replication of sars-cov cc , cytotoxic concentration of the compound that reduced cell viability to %. mean (standard error) of three assays was calculated for each drug. ( figs. a and b and ) . these results indicate that the target(s) of nelfinavir may be involved in the post-entry step of sars-cov replication. to investigate whether or not nelfinavir can affect the efficiency of virion entry, we quantified the copy number of sars-cov rna in vero cells immediately after the entry of virions. real-time rt-pcr revealed that nelfinavir did not affect the entry step of sars-cov infection (fig. ) , which is consistent with our assumption that nelfinavir blocks the post-entry step of sars-cov replication. the mechanisms that underlie the inhibitory action of nelfinavir on sars-cov replication remain to be identified. the main proteinase of sars-cov is one of the molecules expressed after infection with its important role in viral replication [ ] [ ] [ ] , and the effect of nelfinavir on the main proteinase activity should be investigated. we have cloned, expressed, and purified sars-cov main proteinase in order to examine the effect of nelfinavir on this enzyme. our preliminary study indicated that the activity of the main proteinase was blocked only partially (data not shown), which implies that nelfinavir may interact with some molecule(s) other than the main proteinase to fully inhibit sars-cov replication. nelfinavir is a very safe and widely used inhibitor of the hiv- protease, with strong in vivo activity in hiv-infected patients. nelfinavir is generally used in combination with other antiretroviral medications as part of a highly active antiretroviral regimen (haart) [ ] . when used in this manner, - % of patients who are naive to antiretroviral therapy have plasma hiv rna levels below the limit of detection in association with an approximate increase of mm À cd (+) lymphocytes at months of therapy [ ] [ ] [ ] [ ] . the most common side effect of nelfinavir is mild diarrhea, which is observed in - % of patients [ ] . nelfinavir is well tolerated by patients with hiv infection. due to these characteristics, nelfinavir has become one of the most frequently prescribed first line protease inhibitors in the treatment of hiv-infected individuals. our studies have clearly shown that nelfinavir can strongly inhibit the replication of sars-cov in vero e cells. the safety of this drug for humans has already been established, which constitutes the advantages of nelfinavir even for the clinical use to sars patients. our results suggest that nelfinavir should be examined clinically for the treatment of sars. moreover, nelfinavir might be a promising lead compound for anti-sars drugs. a major outbreak of severe acute respiratory syndrome in hong kong national microbiology laboratory, t. canadian severe acute respiratory syndrome study, identification of severe acute respiratory syndrome in canada a cluster of cases of severe acute respiratory syndrome in hong kong epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome aetiology: koch's postulates fulfilled for sars virus effects of a sars-associated coronavirus vaccine in monkeys development of a standard treatment protocol for severe acute respiratory syndrome clinical presentations and outcome of severe acute respiratory syndrome in children antiviral treatment of sars: can we draw any conclusions? clinical features and short-term outcomes of patients with sars in the greater toronto area treatment of sars with human interferons interferon alfacon- plus corticosteroids in severe acute respiratory syndrome: a preliminary study pegylated interferon-alpha protects type pneumocytes against sars coronavirus infection in macaques glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus mechanisms and enzymes involved in sars coronavirus genome expression characterization of a novel coronavirus associated with severe acute respiratory syndrome protease inhibitors: a therapeutic breakthrough for the treatment of patients with human immunodeficiency virus virological and immunological responses to haart in asymptomatic therapy-naive hiv- -infected subjects according to cd cell count a randomized trial comparing initial haart regimens of nelfinavir/nevirapine and ritonavir/saquinavir in combination with two nucleoside reverse transcriptase inhibitors prospective comparison of first-line nelfinavir therapy versus nelfinavir introduction in rescue antiretroviral regimens long-term kinetics of t cell production in hiv-infected subjects treated with highly active antiretroviral therapy viracept (nelfinavir mesylate, ag ): a potent, orally bioavailable inhibitor of hiv- protease this work was supported by grants from the ministry of education, science and culture and the ministry of health, labor and welfare of japan. key: cord- - juhmjaj authors: hou, wei; liu, fei; van der poel, wim h.m.; hulst, marcel m. title: rapid host response to an infection with coronavirus. study of transcriptional responses with porcine epidemic diarrhea virus date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: juhmjaj the transcriptional response in vero cells (atcc® ccl- ) infected with the coronavirus porcine epidemic diarrhea virus (pedv) was measured by rnaseq analysis and hours after infection. differential expressed genes (degs) in pedv infected cells were compared to degs responding in vero cells infected with mammalian orthoreovirus (mrv). functional analysis of mrv and pedv degs showed that mrv increased the expression level of several cytokines and chemokines (e.g. il , cxcl , il a, cxcl [alias il ]) and antiviral genes (e.g. ifi , ifit , mx , oasl), whereas for pedv no enhanced expression was observed for these “hallmark” antiviral and immune effector genes. pathway and gene ontology “enrichment analysis” revealed that pedv infection did not stimulate expression of genes able to activate an acquired immune response, whereas mrv did so within h. instead, pedv down-regulated the expression of a set of zinc finger proteins with putative antiviral activity and enhanced the expression of the transmembrane serine protease gene tmprss (alias mspl) to support its own infection by virus-cell membrane fusion (shi et al, , viruses, ( ): ). pedv also down-regulated expression of ectodysplasin a, a cytokine of the tnf-family able to activate the canonical nfkb-pathway responsible for transcription of inflammatory genes like il b, tnf, cxcl and ptgs . the only cytokine genes found up-regulated by pedv were cardiotrophin- , an il -type cytokine with pleiotropic functions on different tissues and types of cells, and endothelin , a neuroactive peptide with vasoconstrictive properties. furthermore, by comprehensive datamining in biological and chemical databases and consulting related literature we identified sets of pedv-response genes with potential to influence i) the metabolism of biogenic amines (e.g. histamine), ii) the formation of cilia and “synaptic clefts” between cells, iii) epithelial mucus production, iv) platelets activation, and v) physiological processes in the body regulated by androgenic hormones (like blood pressure, salt/water balance and energy homeostasis). the information in this study describing a “very early” response of epithelial cells to an infection with a coronavirus may provide pharmacologists, immunological and medical specialists additional insights in the underlying mechanisms of coronavirus associated severe clinical symptoms including those induced by sars-cov- . this may help them to fine-tune therapeutic treatments and apply specific approved drugs to treat covid- patients. the lack of knowledge for treating hospitalized sars-cov- infected patients is one of the pressing problems of the current covid- pandemic. the sars-cov- virus shows a close genetic similarity to the in april identified sars virus (sars-cov- ) and to other sars-related coronaviruses isolated from humans and bats. sars-cov- induces clinical respiratory symptoms familiar to the virus, mostly in persons with underlying diseases like copd, heart failure, diabetes and obesity ( : wu et al. ) . despite the sars-cov- virus has been extensively studied in the last two decades, there are no vaccines available yet, neither there are effective prophylactic and therapeutic treatment regimens with drugs that work equally well for each individual patient with sars-induced respiratory problems. such treatments might prevent development of severe disease patterns like "acute respiratory distress syndrome" (ards) and other, often fatal complications, and may decrease the case-fatality rate of sars-cov- infections. in our lab we study the alpha-coronavirus pedv. pedv was first detected in pig herds in in europe ( : pensaert and de bouck ) . however, this virus reemerged in the spring of in north america causing a massive outbreak among pig herds, resulting in the death of about % of the suckling piglets due to severe diarrhea and dehydration ( although several studies concluded that these clinical symptoms were caused by mrv itself, in concordance with the co-existence of mrv in pedv infected piglets, also other mrv serotypes were isolated from hospitalized patients with airway problems diagnosed positive for sars-cov- ( : cheng et al. , : duan et al. , : zuo et al. . recently, a cross-family recombinant coronavirus was isolated in china from bat faeces in which an rna sequence originating from the s segment of mrv was inserted in the coronavirus genome between the n and ns a genes, indicating that both viruses were replicating simultaneously in a single cell in bats ( : huang et al. ) . a prevalence study showed that this cross-family recombinant coronavirus circulated in an isolated bat colony in a cave in china ( : obameso et al. ). this cooccurrence of mrv with coronaviruses raised the questions whether a synergistic effect between both viruses exists and if such coexistence plays a role in viral pathogenesis. therefore we studied the host response in cultured cells early ( and hours) after pedv and mrv infection using rnaseq. our original goal was to identify early factors and processes induced by pedv or mrv that could stimulate or influence the replication and pathogenesis of the other virus. the host, tissue and cell tropism of pedv differs from sars-cov- and - . however, the genomic organization, replication strategy and function of a part of the viral nonstructural proteins share common features among all coronaviruses ( : brian and baric ) . this applies particularly for interactions in infected cells of nonstructural coronavirus proteins with specific host proteins. host proteins that are recruited or silenced to support virus replication, assembly and release. in our experiment we used vero cells (cercopithecus aethiops epithelial kidney cell line; atcc® ccl- ) because these cells support efficient infection and replication of both mrv and pedv. vero cells are susceptible for many coronaviruses, including sars-cov- and - ( : chu et al. ). they originate from epithelial tissue, in part resembling nasal and bronchial epithelium cells, the prime target cells infected by sars-cov- in the airways of humans. recent research showed that sars-cov- is also able to replicate in epithelial cells of human small intestinal organoids ( : lamers et al. ) . a disadvantage of vero cells is a deletion in the type i interferon (ifn) gene cluster on chromosome ( : osada et al. ). therefore, these cells lack expression of type i ifns important for activation of antiviral defense mechanisms. however, research has shown that vero cells by-pass this ifnactivation route and could mount an antiviral response mediated by interferon regulatory factor ( : chew et al. ). single infections with pedv or mrv alone and simultaneous (double) infections of vero cells with both viruses were performed using a maximum multiplicity of infection (moi) to achieve a synchronized infection of all cells. by rnaseq measured expression levels of mrna transcripts/genes in infected cells were compared to rnaseq profiles measured from similar treated mock-infected cells harvested at the same time point after infection. the detected sets of differential expressed genes (degs) for pedv and mrv were analyzed by gene set enrichment analysis (gsea) using functional bioinformatic programs to retrieve biological processes (pathways and gene ontology terms [go-term]) and associations with chemical compounds, including drugs. in addition, we searched the literature for functional information of the pedv-degs to find possible associations with sars-cov- pathogenesis. because of the covid- pandemic we gave priority to publish the results of this functional bioinformatical analysis and datamining for the single infected vero cells with pedv separate from the results of the double infections with mrv . in this report we focused on the "very early" host response of epithelial cells to an infection with the coronavirus pedv and pay less attention to the role of specific viral proteins in this host response to pedv. in part our results were in agreement with results of a previous rnaseq study comparing sars-cov- and influenza host responses by rnaseq ( : blanco-melo et al. ). but we also found associations with biological processes, and pivotal genes/proteins acting in these processes, that had not been recognized before. this information may contribute to the search for novel or alternative preventive or therapeutic drugs and treatment protocols for this devastating covid- disease. a time-dependent infection experiment was performed with cultured vero cells. details are described in supplementary file (material and methods) and visually displayed in this file. briefly, overnight cultured vero cells grown in cm wells were mock-infected, infected with mrv strain wbvr ( : hulst et al. ) or pedv strain cv ( : pensaert and de bouck , : rasmussen et al. ] ) with a multiplicity of infection of ≥ for min at °c. for pedv and corresponding mockinfected cells, µg/ml of trypsin in serum-free medium was used to facilitate infection of vero cells during the whole experiment. all virus and mock-infected timepoints were performed in quadruplicate. after incubation for min at °c, virus was discarded and cells were washed twice and supplied with fresh culture medium. cells were incubated for , , , , and h at °c and % co . after incubation for the indicated times, cells were placed on ice before total rna was isolated from three of the quadruplicate wells. the replication of both viruses in vero cells was monitored using virus-specific rt-qpcr tests ( fig. : methods and primers used for pcr are provided in supplementary file ). in addition, cells in one of the quadruplicate wells incubated for h were fixated and stained with antibodies directed against the s spike protein of pedv and the s attachment protein (α ) of mrv . a decrease in ct-values for pedv was not observed before h post inoculation ( h.p.i), indicating that replication in pedv infected cells started later than was observed for mrv (at h.p.i). staining of the cells after h indicated that nearly all vero cells were infected with mrv and more than % with pedv. also more than % of the cells in hwells appeared as fused cells (syncytia), confirming that more than % of the cells were infected with pedv. quality control of the total rna isolated from infected cells using an agilent bioanalyzer showed that rnas isolated from pedv infected wells at h.p.i. were partially degraded (rin values below ), making them unsuitable for rnaseq analysis. therefore, only , and h timepoints were analyzed using rnaseq. stained with a monoclonal antibody directed against the s spike protein. mrv and mock infected cells were stained with a polyclonal rabbit serum raised against a peptide sequence of the s -attachment protein of mrv serotype . nuclei were stained blue with the hoechst, ', -diamidino- -phenylindole dye. equal amounts of total rna isolated from triplicate wells were pooled and subjected to rnaseq analysis by genomescan b.v.(leiden, the netherlands) using next generation sequencing (ngs) (see supplementary file a for details). mapping of ngs reads to the cercopithecus aethiops reference genome and preparation of datafiles with calculated fold change (fc) of expression levels of mapped mrnas, were performed for each comparison at , , and h by genomescan (see supplementary file b). from these datafiles we extracted lists of degs with a fc> and p-value of < . . after accessing the ncbi, panther or kegg databases for human orthologs, not annotated cercopithecus aethiops degs were annotated with an hugo official gene symbols (http://www.genenames.org). in supplementary file sheet "pedv-mrv degs fc> ", lists of all annotated pedv and mrv degs are presented with their fc. in a separate sheet "pedv-degs functional info" all individual degs regulated by pedv at and h.p.i. are presented with their fc, information about their function and the types of human cells in which expression of the gene is relatively high compared to other human cells (retrieved from the "primary cell atlas" dataset of biogps: http://biogps.org/). note that all tables in these excel sheets of supplementary file are sortable using the headers. in all results paragraphs beneath information about the biological function of degs was retrieved by consulting the "genecards" (weizmann institute of science: https://www.genecards.org/) and ncbi gene reports (entrez gene: https://www.ncbi.nlm.nih.gov/gene/), and literature linked to these reports (for references about these biological functions of genes/proteins we refer to publications cited in these reports: "genecards" weblinks are provided in supplementary file ). sets of pedv and mrv degs were analyzed using the gsea program geneanalytics (lifemap sciences, inc.) and pathways (for mrv and pedv), go-terms (not for mrv), and associations with compounds/drugs (not for mrv) with a high or medium score (p-value < . ) were retrieved and listed in separate sheets in supplementary file (sheets "mrv-pedv pathways", "pedv g -terms" and "pedv compounds"). similar and related pathways retrieved for both pedv and mrv, and remarkable pedv pathways, go-terms and compound associations are summarized in table . for pedv all degs within these pathways are provided with their regulation, up (green) or down (red). for mrv only degs in common with pedv-degs were listed in table (see sheet "mrv-pedv pathways" in supplementary file for all mrv-degs acting in these pathways). subsets of pedv-degs were selected matching the terms "chemokines-cytokines", "antiviral" , and terms related to the pathogenesis of covid- (explained below) using the genotyping program varelect (lifemap sciences, inc.) and displayed in supplementary file in separate sheets: "chemokines-cytokines", "(anti)-viral", etc. based on these selections we prepared a set of pedv key-degs consisting of genes regulated with a fc of > (up) or <- (down) or playing an important role in biological processes induced by pedv and related to covid- pathology. in beneath results sections we tried to give as much as possible meaningful information about the function of key-degs for which we found an association with sars-cov- infections. we emphasize that further dedicated experimental and in-silico research is necessary to confirm the involvement of the proteins encoded by these genes for pathogenesis of this viral disease. table . enriched pathways, go-terms and compound associations of pedv-degs. *pedv enriched pathways (a), go-terms (b), and associations with compounds and drugs (c) with a high and medium score and with at least matching genes were retrieved from geneanalytics. common pathways for mrv were included in table a . a full list of pathways with degs, retrieved for mrv at and h, is provided in supplementary file (sheet mrv-pedv pathways). a possible function or process related to specific degs, pathways, go-term, or compounds/drugs is provided in blue text between brackets. $ official gene-symbols (hugo abbreviations) are listed for degs. down-regulated degs were colored red and up-regulated degs were colored green. in section a the number of degs regulated by mrv in a pathway and the common degs are provided between brackets. degs regulated by both pedv and mrv are underlined. compared to mrv, only a few genes involved in "cytokine/chemokine signaling" were regulated at and h by pedv. in fig. a the regulation of cytokines/chemokines in pedv and mrv infected vero cells are displayed. this indicated that mrv increased the transcription of a broad set of cytokines/chemokines, including interferonmediated cytokines like cxcl and cxcl (alias il ), already at h.p.i., whereas pedv did not, even not when replication of pedv rna was detected by rt-qpcr at h.p.i.. for mrv, this cytokine/chemokine response at h.p.i. was followed by high up-regulation of "hallmark" antiviral genes at h.p.i. (see fig. b : e.g. interferoninduced genes [ifi] and oasl) and chemokines that attract t cells, monocytes, granulocytes, including basophils (e.g. cxcl , cxcl and ccl ). pedv infection up-regulated only a few genes coding for proteins with cytokine activity (ctf and edn ), and also did not elevated gene expression of these "hallmark" antiviral genes. in contrast, pedv down-regulated expression of genes (out of in total) coding for zinc finger proteins (out of in total), all with an antiviral activity towards herpes simplex virus ( fig c) binding of thrombin to f rl reduces inflammation, activates platelets and increases vasodilation and permeability of the vascular wall (see also below in the section "platelets activation"). csf r is a receptor for the cytokine colony stimulating factor , a cytokine that regulates differentiation and function of macrophages, and in the cns, the density and distribution of microglia cells. the blnk gene codes for a cytosolic protein that passes on b-cell receptor signals in the signaling cascade that activates b-cell development and function. gene expression of genes coding for essential components of this b-cell signaling, like "spleen associated tyrosine kinase" (syk) and "lyn proto-oncogene"(lyn) were not regulated by pedv, nor by mrv. genes involved in amino acid, protein translation and metabolism of immuno-active compounds. pedv degs coding for enzymes involved in the metabolism of the non-essential amino acids histidine, phenylalanine, tryptophan and proline were found enriched in the pedv dataset (see supplementary file , sheet pedv-compounds). remarkable were the degs coding for amine oxidases involved in the catabolism of the biogenic amines histamine, tryptamine and phenylethylamine, their derivates and related substrates/products of these enzymes (fig. , aoc , maoa, il i ). none of these amino oxidase genes were regulated by mrv. using the genotyping program varelect, pedv-degs with an association with these biogenic amines were retrieved (supplementary file , sheet "biogenic amines"). three enzymes clustered in the "histidine metabolism" pathway (https://www.kegg.jp/keggbin/show_pathway?hsa + ) with histamine and reaction products generated from this biogenic amine (fig. ) . also most association of pedv-degs were found by varelect for histamine. the gene coding for the amine oxidase "interleukin induced " (il i ) was strongly down-regulated ( -fold) h after infection with pedv. besides catalysis of l-phenylaniline into phenylpyruvate (fig. ) , il i also fulfills an important role in signaling in "synaptic clefts" formed between antigen presenting cells (apc) and t cells (so-called "immune cleft": see also below) ( expression of the prostaglandin-endoperoxide synthase (ptgs ) gene was upregulated by mrv at h p.i. ptgs synthesizes prostaglandin endoperoxide h (pgh ), an compound with a short half-life and the precursor of many biological active prostaglandins: e.g. thromboxane-a (mediates activation of platelets), pgi and pge . in contrast, pedv increased the expression of the gene coding for prostaglandin e synthase (ptges) which converts pgh into pge . pge is a direct vasodilator, but does not inhibit platelet aggregation. pge also suppresses t cell receptor signaling. pedv decreased expression of the gamma-glutamyltransferase gene (ggt ) after h ( -fold), but increased expression of this gene hours later to a -fold level compared to mock infected cells. ggt synthesizes leukotriene d (ltd ) from ltc . ige-activated mast cells may secrete ltd and ltc , together with histamine and platelets activating factor (paf). this vesicle mediated secretion by mast cells (degranulation) results in stimulation of mucus production, and similar to histamine, increases the permeability and smooth muscle contraction of the vascular wall. in persons suffering from asthma this degranulation leads to an immediate allergic response (bronchospasm, airflow obstruction and forming of edema). genes involved in "cilia and synaptic cleft" formation. gsea detected "axon guidance" as the go-term with the highest score for pedv (see table ). in addition, pedv-degs were enriched coding for proteins involved in calcium ion-dependent exocytosis from vesicles into the "synaptic clefts" between two cells (e.g. between axons and dendrites), and degs coding for proteins involved in formation of cilia. cilia protruding from cells are found in many forms. they can have a static (structural) function, e.g. in forming of clefts between two cells (see fig. ), or a motile function. motile cilia on the surface of ciliated cells lining up the epithelial layers in the nose, trachea and bronchia sweep out superfluous mucus containing dirt from the airways. pedv degs matching the terms "cilia" and "synaptic cleft" retrieved form the genotyping program varelect were further examined by consulting functional information in the ncbi gene and genecards reports in order to evaluate their association with these processes (see supplementary file , sheet "cilia and synaptic cleft"). based on this analysis we identified genes in the set of pedv degs which can i) negatively regulate cell adhesion (rnd and sema a), ii) inhibit formation of cilia (kinases mak and cdk , highly up-regulated at h), and iii) regulate cytoskeleton rearrangements that facilitate axon growth and growth and stabilization of dendritic spines (f rl , regulation of genes involved in histamine/biogenic amines (see above) and formation of cilia/clefts suggested that gene expression related to this intersynaptic signaling between immune cells could be affected in response to infection with pedv (fig ) . in particular, the highly down-regulated gene il i ( -fold at h p.i.) is of interest (see also above). il i is believed to be secreted from apc's (e.g. dc's) in the immune cleft formed with t cells ( : molinier-frenkel et al. ). the mechanism how il i transmits its signal to t cells is not completely understood. it could bind to a receptor that concentrates this amino oxidase in the cleft, resulting in elevated h o and ammonia production, phenylalanine depletion and phenylpyruvate production in the cleft space. these alteration in the concentration of these chemicals in the cleft are sensed by the t cell. the paralog of il i , amino oxidase maoa (down-regulated -fold by pedv) could also play a similar role in this signaling process. remarkable was also the strong down-regulation of genes coding for the olfactory receptor family subfamily a member (or a ; -fold at h) and anoctamin (ano , alias cacc; -fold at h). ano is a calcium-activated chloride channel imbedded in the basal membranes of neurons that harbor apical membrane receptors like or a that sense odorants. by importing chloride ions into the cytosol ano contributes to the depolarization of these neurons (https://www.kegg.jp/kegg-bin/show_pathway?hsa + ). loss of smell and taste is one of the first noticeable symptoms of covid- . genetic defects in the ano gene are associated with von willebrand disease, a bleeding disorder due to defective platelet aggregation ( . also disease incidence in adult males is significantly higher than in females of the same age. to assess whether pedv-degs relate to these pathological symptoms, the genotyping program varelect was used to identify genes matching the terms "ards", "cardiomyopathy", "obesity (diabetic)", and "platelets activation". detailed information about all matching degs is provided in supplementary file in separate sheets for all queried terms. degs matching to more than one query term are displayed in fig. . remarkable associations of degs with these terms are mentioned in sections beneath. . edn and agt are vasoactive peptides and binding of edn and agt to their receptors on granular cells of the juxtaglomerular apparatus in the kidney raises free calcium levels in the cytosol, leading to inhibition of camp-mediated secretion of the aspartylprotease renin (ren), the key regulator of renin-angiotensin-aldosterone system (raas) (https://www.kegg.jp/kegg-bin/show_pathway?hsa + ). ren converts pre-angiotensinogen (agt) to the endocrine peptide-hormone agt (https://www.kegg.jp/kegg-bin/show_pathway?hsa + ). agt is further cleaved to variants with specific endocrine activity by angiotensin i converting enzymes (e.g. ace and ace : ). on the surface of bronchial epithelial cells ace was identified as entry receptor for sars-cov- and - ( : hoffmann et al. ). the octamer peptide agt stimulates secretion of the mineralocorticoid hormone aldosterone by the adrenal glands. aldosterone, and the agt and edn peptide hormones regulate an array of physiological processes in the body, e.g. vascular smooth muscle contraction, blood pressure, fluid and electrolyte homeostasis ( : agapitov et al. ) . all processes that are important for proper functioning of the vascular system, heart muscles and kidneys. ctf is directly involved in the pathology of numerous cardiovascular diseases by promoting cardiac myocyte hypertrophy ( : wollert et al. ) , which may lead to the onset of heart-diseases like "hypertrophic cardiomyopathy" or "dilated cardiomyopathy", and eventually, to (lethal) heart failure. pedv induced a strong down-or up-regulation of several other genes directly involved in the function of cardiomyocytes. sodium voltage-gated channel subunit (scn b) was strongly upregulated ( -fold) and mylk (see above), citrate synthase (cs; down-regulation -fold) and ankyrin repeat domain (ankrd ) were strongly down-regulated. down-regulation of cs may reduce oxidative capacity in cardiomyocytes. gene expression of ankrd was down-regulated -fold in response to mrv infection at h.p.i, but reverted to a -fold up-regulation h later. ankrd is a putative transcription factor involved in regulation of gene expression in hypertrophic myocytes (https://www.wikipathways.org/index.php/pathway:wp ) regulation of cytosolic calcium levels in the cytosol of cardiomyocytes, e.g. by binding of col a and col a (up-regulated by pedv, see above) to integrin subunit alpha on the surface of cardiomyocytes or after import of calcium ions mediated by calcium voltage-gated channels (e.g. by cacna h; down-regulated -fold at h by pedv) may also trigger myocyte hypertrophy. pedv strongly down-regulated gene expression of a potassium voltage-gated channel (kcnq ; -fold). this in contrast to a strong up-regulation of the sodium symporter scn b. for the potassium channel cacna h (alias kv . ) it was reported that this channel regulates the membrane potential and ca + permeability of mitochondria located in the vicinity the sarcoplasmic reticulum in rat cardiomyocytes ( : testai et al. ). all three above mentioned ion channels are also involved in the process of excitation, contraction/relaxation and repolarization of cardiac myocytes. mrv also downregulated gene expression to -fold for the potassium (kcnq ) and calcium channel cacna h, but did not increased expression of the sodium symporter gene scn b or orthologs of this gene. chronic hypertension and heart disease/failure is a complication frequently observed in obese/diabetic patients. in accordance with this, out of the pedv degs matching the term "obesity" also matched with the term "cardiomyopathy" (see additional remarkable pedv-degs. highly up-or down-regulated pedv-degs not mentioned in the text, and to our opinion interesting with regard to coronavirus infection, are briefly described in table . among these degs several genes coding for transcription factors and genes transcribed in antisense rna's that inhibit translation of their coding counterparts. for more functional information about these degs we refer to the weblinks provided in supplementary file , sheet "pedv key degs". table . remarkable pedv-degs not mentioned in the text. in this report we measured the transcriptional response of vero cells shortly after infection with the coronavirus pedv. the function of the responding host genes and the biological processes in which they act were studied in detail by us to find plausible relations to covid- pathology. because of the differences in genomic organization and expression of viral proteins between sars-cov- and pedv, we paid less attention to couple the response of specific host genes to the function of specific coronavirus proteins. we were able to infect the majority of vero cells (> %) with pedv and mrv synchronically. this resulted in a unique set of highly up-and down-regulated degs for pedv. not more than % of the pedv-degs (n= ) were similar to mrv-degs (total of mrv-degs). in contrast to mrv, we observed no typical response of antiviral genes and related cytokine/chemokine genes in vero cells within h.p.i. for pedv. for mrv these processes started already before h.p.i.. we have to notice that pedv replication started h later than mrv replication, which could in part be the reason for not detecting transcriptional regulation of specific cytokine, chemokine and antiviral genes for pedv. longer incubation times than h were not planned in the original design of our experiments and would have resulted in a set of pedv-degs dominated by genes involved in syncytia forming and apoptotic/necrotic cell death. nevertheless, at h replication of pedv rna was detected by rt-pcr, indicating that dsrna was present in the cells and could have be sensed by cytosolic pattern recognition receptors of the rig-i-like family to initiate an antiviral and related cytokine/chemokine response. similar as observed in another study with pedv and vero cells, and in analogy with sars-cov- and - , we observed a high up-regulation of the transmembrane serine protease gene (tmprss ) that acts as a co-factor in the infection process of cells ( we observed a reduced expression of eef a , as part of a transcription factor-complex that binds and activates the promotor of ifnγ, and of the cytokine eda and its receptor (edaradd) involved in activation of canonical-nfkb transcription of antiviral cytokine-chemokine genes like cxcl (alias il ) an cxcl . this reduced expression of eef a and eda and its receptor may play a crucial role in delaying or downgrading an ifn-mediated antiviral and cytokine/chemokine response in our vero cell system. the elevated transcription of many cytokine and chemokine genes in vero cells by mrv suggests that replication of this rna virus in epithelial cells induces secretion of these immune effectors (more information about the genes/processes that responded to mrv infection will be published elsewhere). pedv, and also the mrv strain we used both replicate in enterocytes lined up in the intestinal mucosal layer. in the intestinal and bronchial epithelial layer, microfold (m) cells are imbedded between these lined up epithelial cells. m-cells sense and engulf foreign pathogens/antigens from the lumen to present them to residing immune cells. according to pathway analysis, most t cell related immune genes regulated in response to mrv infection were part of the pathways "t-helper (th ) differentiation/activation" and "il signaling". antigen presentation by m cells to th cells in the submucosal layers stimulate secretion of different types of il cytokines (il a-d, il and il f) resulting in activation of different types of innate immune cells and t cells, including th /th cells. dysregulation of this pathogen-induced il response may disturb the balance between th /th -cell mediated immune responses, resulting in excessive inflammation, damage to the epithelial layer and on the long term, to autoimmune reactions. tf rorc (or specific isoforms of this tf, see above) plays a pivotal in controlling il expression and secretion by th cells. pedv strongly up-regulated gene expression of rorc in vero cells whereas mrv down-regulated expression of this tf. this difference in regulation of tf rorc suggests that virus-induced activation or suppression of il secretion by th cells in submucosal layers of airway epithelium can be an important mechanism to dysregulate the activation of t cell responses. therefore, tf rorc might be a potential target for drug treatment/development. drugs affecting expression of rorc, like the fluorinated steroid "dexamethasone" and the synthetic tetracycline derivative "doxycycline" (http://ctdbase.org/basicquery.go?bqcat=gene&bq=rar+related+orphan+receptor +c) are already under investigation in relation to sars-cov- pathogenesis. transcriptional regulation of a set of genes coding for enzymes involved in biogenic amine metabolism was unique for pedv, and not observed for mrv. most associations of these pedv-degs were found with histamine, a compound produced by mast cells and basophils, and released by these cells in response to allergens and pathogen-induced inflammation. the -fold up-regulation of the enzyme aoc suggests that histamine is converted to imidazole-acetaldehyde (see fig. ). however, without data of intra-and extracellular concentrations of the chemicals this remains speculative. recent reports indicated that submucosal mast cells in the lungs were triggered by sars-cov- infection to release pro-inflammatory cytokines (il , il and tnf-alpha) . mdscs infiltrate these tumors and inflamed tissues to suppress the local activity of specific immune cells. therefore, the role of infiltrating mdscs at inflammatory sites in the lungs of covid- patients, as part of an sars-cov- immune-evading strategy, and the role of il i in this process, is worthwhile to investigate in more detail. expression of genes that promote or inhibit the formation and motility function of cilia were time-dependently regulated by pedv. the -fold up-regulation of fez gene expression at h ( -fold) descended within h to a moderate -fold upregulation. this descend occurred simultaneously with elevation of gene expression of the kinases mak and cdk , both involved in inhibition of cilia formation. because pedv efficiently replicates in enterocytes that carry ciliated membrane protrusions (microvilli) on their luminal surface, regulation of these genes could be related to structural changes in the cytoskeleton of cells imposed by virus replication (e.g. syncytia forming in pedv infected vero cultures). likewise, sars-cov- replication could also impose structural changes in cilia protruding from the surface of upperairway epithelial cells (nose, trachea) and bronchi. based on our results we cannot pinpoint a specific process in which these cilia-regulating genes act. possible processes can either be formation of an immune cleft, a virological cleft to promote more effective infection of neighboring cells, or cytoskeleton rearrangements to support virus replication, morphogenesis and budding from cells. interestingly, two recent studies revealed a high level of expression of the sars-cov- entry receptor ace and its co-receptor tmprss in ciliated airway epithelial cells ( . these processes deserve more attention, and may also be considered as possible target-processes for interference with drugs. within our set of pedv-degs, and the biological processes deduced from this set, we found associations with diverse aspects of covid- pathogenesis, i.e. "ards", "cardiomyopathy", "obesity (diabetic)", and "platelets activation". however, it is unknown whether the proteins encoded by these pedv-degs indeed play a role in the biological processes underlying the symptoms and complications observed in hospitalized persons infected with sars-cov- . nevertheless, a part of these genes/processes may be starting points for further dedicated research. research to fine tune drug treatment protocols that are already applied for covid- patients, or research that provides new insights for treatments with alternative prophylactic and therapeutic approved drugs. in table . we summarized the pedv-degs that are, to our opinion, of interest for modulation of the biological processes underlying the pathogenesis of covid- . table . possible target genes for covid- therapy. colophon. the overwhelming amount of data published recently made it impossible for us to oversee all (novel) facts about the sars-cov- virus and pathology of the covid- disease. some important genes and related processes imbedded in our set of pedv-degs may have been overlooked by us. therefore, we encourage researchers, especially medical, immunological and pharmaceutical specialists, to study this set of degs in detail. the users-friendly supplementary file with functional information about degs and related biological processes can be down-loaded from the web. by publishing these pedv data ahead of our complete study, we hope that some of the gene targets and cognate processes we have identified for the coronavirus pedv will contribute to a better understanding how hospitalized covid- patients can be treated and cured. a more condensed version of this manuscript, focusing on the original goal of our study, will be submitted to a peer-reviewed virological journal soon. risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease a new coronavirus-like particle associated with diarrhea in swine origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis a novel pathogenic mammalian orthoreovirus from diarrheic pigs and swine blood meal in the united states identification of a novel reassortant of a mammalian orthoreovirus in faeces of diarrheic pigs in the netherlands: presentation at the th annual meeting of epizone in paris high similarity of novel orthoreovirus detected in a child hospitalized with acute gastroenteritis to mammalian orthoreoviruses found in bats in europe pathogenesis of reovirus infections of the central nervous system identification and characterization of a new orthoreovirus from patients with acute respiratory infections a novel reovirus isolated from a patient with acute respiratory disease reov isolated from sars patients cloning and identification of reovirus isolated from specimens of sars patients a bat-derived putative cross-family recombinant coronavirus with a reovirus gene the persistent prevalence and evolution of crossfamily recombinant coronavirus gccdc among a bat population: a two-year followup coronavirus genome structure and replication comparative tropism, replication kinetics, and cell damage profiling of sars-cov- and sars-cov with implications for clinical manifestations, transmissibility, and laboratory studies of covid- : an observational study sars-cov- productively infects human gut enterocytes the genome landscape of the african green monkey kidney-derived vero cell line characterization of the interferon regulatory factor -mediated antiviral response in a cell line deficient for ifn production imbalanced host response to sars-cov- drives development of covid- full-length genome sequences of porcine epidemic diarrhoea virus strain cv ; use of ngs to analyse genomic and sub-genomic rnas tmprss and mspl facilitate trypsin-independent porcine epidemic diarrhea virus replication in vero cells desc and mspl activate influenza a viruses and emerging coronaviruses for host cell entry efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss a novel isoform of the orphan receptor rorγt suppresses il- production in human t cells structure, function and evolution of the hemagglutinin-esterase proteins of corona-and toroviruses functional screen reveals sars coronavirus nonstructural protein nsp as a novel cap n methyltransferase '-o methylation of the viral mrna cap evades host restriction by ifit family members human il i is a secreted lphenylalanine oxidase expressed by mature dendritic cells that inhibits t-lymphocyte proliferation the il i enzyme: a new player in the immunosuppressive tumor microenvironment avoiding the void: cell-to-cell spread of human viruses a common -kb deletion involving vwf and tmem b in german and italian patients with severe von willebrand disease type pathological findings of covid- associated with acute respiratory distress syndrome case-fatality rate and characteristics of patients dying in relation to covid- in italy the tn antigen-structural simplicity and biological complexity post-translational modifications of coronavirus proteins: roles and function emerging wuhan (covid- ) coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human cd therapies for bleomycin induced lung fibrosis through regulation of tgf-beta induced collagen gene expression covid- -induced acute respiratory failure: an exacerbation of organspecific autoimmunity? medrxiv . cardiotrophin- activates a distinct form of cardiac muscle cell hypertrophy. assembly of sarcomeric units in series via gp /leukemia inhibitory factor receptor-dependent pathways a g polymorphism of the endothelin- gene and atrial fibrillation in patients with hypertrophic cardiomyopathy sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor role of endothelin in cardiovascular disease expression and function of kv . channels in rat cardiac mitochondria: possible targets for cardioprotection human coronary arteriolar dilation to adrenomedullin: role of nitric oxide and k(+) channels adrenomedullin improves cardiac function and prevents renal damage in streptozotocin-induced diabetic rats adrenomedullin: a possible autocrine or paracrine inhibitor of hypertrophy of cardiomyocytes histones and heart failure in diabetes histone h lysine hypoacetylation is associated with defective dna repair and premature senescence in zmpste -deficient mice studies of the sim gene in relation to human obesity and obesity-related traits neuropeptide b mediates female sexual receptivity in medaka fish, acting in a female-specific but reversible manner porcine epidemic diarrhea virus inhibits dsrna-induced interferon-β production in porcine intestinal epithelial cells by blockade of the rig-imediated pathway mast cells contribute to coronavirus-induced inflammation: new anti-inflammatory strategy mast cell stabilisers, leukotriene antagonists and antihistamines: a rapid review of effectiveness in covid- lnc-c/ebpβ modulates differentiation of mdscs through downregulating il i with c/ebpβ lip and wdr identification of discrete tumor-induced myeloid-derived suppressor cell subpopulations with distinct t cellsuppressive activity sars-cov- entry factors are highly expressed in nasal epithelial cells together with innate immune genes sars-cov- receptor ace and tmprss are primarily expressed in bronchial transient secretory cells mechanisms of innate immune evasion in re-emerging rna viruses supplementary file : interactive excel file with sortable tables in separate sheets. please, first read the sheet "read me" for an explanation and instructions for the use of the tables. excel sheets contain tables with i) pedv and mrv degs extracted from rnaseq data files, ii) functional information about the pedv-degs, iii) gsea extracted pathways (for mrv and pedv), go-terms (only for pedv) and compound associations (only for pedv), and iv) associations of pedv-degs with the terms "cytokines-chemokines", "(anti)-viral", "biogenic amines", "cilia and synaptic clefts", and the disorders "ards, "cardiomyopathy", "obesity", and "platelets activation" (a-c-o-p). the authors declare that they have no competing interests.authors' contributions. key: cord- -lvxxdvas authors: shan, dan; fang, shouguo; han, zongxi; ai, hui; zhao, wenjun; chen, yuqiu; jiang, lei; liu, shengwang title: effects of hypervariable regions in spike protein on pathogenicity, tropism, and serotypes of infectious bronchitis virus date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: lvxxdvas to study the roles of hypervariable regions (hvrs) in receptor-binding subunit s of the spike protein, we manipulated the genome of the ibv beaudette strain using a reverse genetics system to construct seven recombinant strains by separately or simultaneously replacing the three hvrs of the beaudette strain with the corresponding fragments from a qx-like nephropathogenic isolate ck/ch/ldl/ from china. we characterized the growth properties of these recombinant ibvs in vero cells and embryonated eggs, and their pathogenicity, tropism, and serotypes in specific pathogen-free (spf) chickens. all seven recombinant ibvs proliferated in vero cells, but the heterogenous hvrs could reduce their capacity for adsorption during in vitro infection. the recombinant ibvs did not significantly increase the pathogenicity compared with the beaudette strain in spf chickens, and they still shared the same serotype as the beaudette strain, but the antigenic relatedness values between the recombinant strain and beaudette strain generally decreased with the increase in the number of the hvrs exchanged. the results of this study demonstrate the functions of hvrs and they may help to develop a vaccine candidate, as well as providing insights into the prevention and control of ibv. ibv primarily replicates in the epithelial surface of the respiratory tract, although some strains are nephropathogenic (cavanagh, ) . ibv infections reduce egg production, quality, and hatchability, as well as increasing the feed conversion ratio and carcass condemnation in slaughterhouses. thus, infectious bronchitis causes severe economic losses in the poultry industry worldwide. the enveloped ibv belongs to the genus coronavirus, family coronaviridae, order nidovirales (cavanagh, ) . the main structural protein is the spike (s) glycoprotein, which comprises a divergent s subunit and conserved s subunit (de groot et al., ; shil et al., ) . the s subunit contains the receptor-binding domain (wickramasinghe et al., ) , and it carries virus-neutralizing and serotype-specific determinants. the s domain exhibits high sequence diversity, where %- % (even up to %) of the amino acids differ within the s subunit among ibv serotypes. after comparing the s subunit from a number of distinct ibv isolates, a previous study defined particularly variable segments at the amino terminus of the s subunit as hypervariable regions (hvrs) . the hvrs possibly account for the antigenicity and serotypic variation, and evidence indicates that five neutralizing peptides mainly mapped onto the s subunit are co-located within the hvrs (cavanagh et al., (cavanagh et al., , moore et al., ; niesters et al., ) . in addition, the hvrs are possibly associated with receptor binding. it has been reported that the critical amino acids for attachment of the m spike overlap with a hvr in the s subunit (promkuntod et al., ) . thus, the hvrs may affect the tropism, serotype, and pathogenicity of ibv, and it is important to elucidate their biological functions. previous studies applied forward genetics methods to determine the roles of the hvrs by characterizing their phylogenetically closely related isolates with mutations in other parts of the ibv genome, but it is difficult to establish a precise model for further analysis based on these studies. thus, in this study, we manipulated the ibv beaudette genome by reverse genetics to exchange the hvrs in the beaudette strain with those in ck/ch/ldl/ in order to precisely determine their biological functions. we also explored the possibility of developing a reverse genetic vaccine candidate cultured in vero cells to provide protection from the prevalent field strains. the results of this study contribute to our understanding of the prevention and control of ibv. vero cells were maintained in dulbecco's modified eagle's medium (gibco, grand island, ny, usa) supplemented with % fetal bovine serum (sigma-aldrich, saint louis, mo, usa), penicillin ( units/ ml), and streptomycin ( μg/ml) at °c with % carbon dioxide. the ck/ch/ldl/ strain was isolated from h -vaccinated chickens with renal lesions in . the beaudette strain was routinely propagated in vero cells (liu et al., , tay et al., ) and the % tissue culture infection dose (tcid ) of the viral stock was calculated using -well plates with -fold serial dilutions. the % egg infection dose (eid ) of the viral stock was determined by inoculating -fold dilutions into groups of -day-old embryonated chicken eggs. five fragments spanning the entire ibv genome were obtained by rt-pcr from vero cells infected with the ibv beaudette strain, as described previously (fang et al., ) . the three hvrs in the isolate ck/ ch/ldl/ were identified based on alignments of the amino acid sequences, as described previously . the sequences of the ibv cdna covering the three hvrs in the s gene were replaced separately or simultaneously with those from ck/ch/ldl/ , and subsequently ligated into the full-length ibv cdna ( fig. a and table ). full-length transcripts generated in vitro were introduced into vero cells by electroporation. ibv n gene transcripts were also generated to enhance the efficiency of viral recovery. total rna was prepared from the electroporated vero cells or infected allantoic liquid. viral rna replication was investigated based on rt-pcr of negativestrand genomic rna (tan et al., ) . the s gene in the recovered ibv clones (third passage in vero cells and embryonated eggs) was amplified by rt-pcr and subsequently confirmed by dna sequencing analysis. the gene was characterized during the third passage of viruses in specific pathogen-free (spf) embryonated eggs or vero cells. the seven rescued recombinant ibvs were designated as rhvr i, rhvr ii, rhvr iii, rhvr i/ii, rhvr i/iii, rhvr ii/iii, and rhvr i/ii/iii. to determine the growth kinetics of the rescued recombinant ibvs, a dose of × eid was inoculated into the allantoic cavities of -dayold embryonated eggs, and the allantoic fluid was harvested from three eggs in each group at , , , , and h post-inoculation, where the fluids from three eggs were pooled for eid determination with three replicates. vero cells were infected with beaudette and recombinant ibvs, and three wells were harvested at , , , , , and h post-infection. viral stocks were prepared by freezing/thawing the cells three times to determine the tcid with three replicates for each time. ibv infected cells cultured in six-well plates were washed with phosphate-buffered saline (pbs), fixed with % paraformaldehyde for min, and permeabilized with . % triton x- for min. if staining was performed with a monoclonal antibody d against ibv n protein and subsequently with fitc-conjugated anti-mouse igg (sigma-aldrich). cells were examined by fluorescence microscopy. all of the animal experimental procedures were approved by the ethical and animal welfare committee of heilongjiang province, china (license no. sq ). we randomly assigned -day-old spf layer chickens to groups, i.e., the seven recombinant strains, beaudette, ck/ch/ldl/ , and pbs groups. there were spf chickens in each group. the challenge strain ( × eid per bird) was applied via the intranasal and ocular routes. the clinical signs were monitored in randomly selected challenged birds from each group, and the morbidity and mortality rates were recorded daily. we evaluated the challenged chickens blindly for respiratory rales at days after challenge. signs were scored as = absent, = mild, = moderate, or = severe. at days after ibv challenge, the other five birds in each group were killed humanely using carbon dioxide over inhalation, followed by exsanguination. the cranial third of the trachea, lungs, kidneys, and cecum tonsils were collected, partly fixed in formalin, and embedded in paraffin. longitudinal -μm sections were stained with hematoxylineosin (h&e stain). the mucosal thickness, deciliation, goblet cells, and lymphocytes scores for the tracheal mucosa were evaluated blindly and scored from to based on their severity (i.e., normal, mild, moderate, marked, and severe) (van ginkel et al., ) . moreover, viral shedding was quantified in the oropharyngeal secretions from infected chickens at , , , , and days after challenge by real-time rt-pcr (jones et al., ) . the tissues collected at days post-ibv challenge, i.e., tracheas, lungs, kidneys, and cecum tonsils, were also subjected to immunohistochemistry (ihc) using monoclonal antibody d , as described previously (de wit et al., ; xu et al., ) . viral loads in selected tissues were determined based on ibv rna detection by realtime rt-pcr, as described previously . the seven ibv recombinant strains, beaudette, and ck/ch/ldl/ were analyzed in cross-virus neutralization tests. sera against the ibv strains were prepared as previously described . in the virus neutralization tests, sera were serially diluted two times with sterile pbs and mixed with × eid or tcid for the ibv strains. the two-way cross-neutralization test between the beaudette and recombinant ibvs was performed in vero cells. after incubation for h at °c, the virus-serum mixtures were cultured in -well microplates for days. we could not detect the replication with ck/ch/ ldl/ in vero cells, so the neutralization tests were performed in -day-old spf embryonated eggs to confirm whether the serotypes of the recombinant ibvs belonged to ck/ch/ldl/ . the end-point titer for each serum sample was calculated using the reed-muench method. antigenic relatedness values were calculated (archetti and horsfall et al., ; wadey and faragher, ) . the celisa protocol used in this study is similar to the conventional elisa. the viral solution for each strain was serially diluted two times with sterile pbs and coated on the plate. standard curves were drawn according to the standard elisa method. vero cells were cultured overnight in -well microplates (corning, usa) ( cells/well). for the adsorption assay, each strain was incubated in cells at a multiplicity of infection (moi) of for h at °c with three replicates (fang et al., ; sun et al., ) . after two washes with pbs, the microplates were fixed with methanol and % hydrogen peroxide for min, and then permeabilized with . % triton x- for min at room temperature. next, the microplates were washed three times and blocked with % skim milk for min at room temperature. after washing three times, the microplates were incubated with the primary mouse monoclonal antibody d for h at °c. a similar procedure was then performed where the plates were incubated for min with the peroxidase-conjugated anti-mouse igg. peroxidase substrate solution (tmb; sigma-aldrich) was added and the plates were developed in darkness. the color reaction was stopped with stop solution (sigma-aldrich) and the absorbance was read at nm. for the internalization assay, cells were seeded into -well microplates and inoculated with each strain for h at °c. after incubation, the cells were washed twice with pbs, before treating the infected cells with citrate buffer for min to inactivate the adsorbed but not internalized virus. the cells were washed with pbs to remove the citrate buffer. the celisa process was performed as described above. the celisa results were expressed as the relative viral load adsorbed or internalized into cells relative to the beaudette control. vero cells were seeded in -well plates (corning) and infected with each separate strain at an moi of . in the adsorption and internalization stage, total rna was extracted from the cells, before storing at − °c for the subsequent quantification of viral loads, as described previously (liu et al., a; sun et al., ) . three replicates were analyzed for each sample. first, the stable expression levels of three candidate reference genes comprising s rna, β-actin, and gapdh were compared by real-time rt-pcr in vero cells infected with the beaudette strain at °c or °c for h. the results were analyzed using genorm software, normfinder software, and delta ct (http:// . . . /referencegene.php). the viral mrna levels of all the samples were calculated by using the most stably expressed genes as an internal reference for normalization (fung et al., ) . the real-time rt-pcr results were expressed as the relative viral load adsorbed or internalized into cells relative to the beaudette control. we randomly assigned -day-old spf layer chickens to groups, with birds in each group. birds in groups - were inoculated with the seven recombinant ibvs, beaudette, and ck/ch/ldl/ ( × eid per bird) via intranasal and ocular routes. birds in group were inoculated with pbs and treated as the negative control. half of the surviving birds in each group were randomly selected and challenged after days with a × eid dose of ck/ch/ldl/ via intranasal and ocular routes. the clinical signs were monitored in the challenged birds in each group, and the morbidity and mortality rates were recorded daily. viral shedding was also quantified using oropharyngeal swabs from the challenged chickens at , , , , and days after challenge by real-time rt-pcr (jones et al., ) . the remaining chickens in each group were investigated for days. were conducted based on three replicates. anova followed tukey's multiple comparison tests were performed to compare the virus titers of different strains at the same time point, and differences were considered significant at p < . . one-way anova followed tukey's multiple comparison tests were used to compare differences in the viral loads in selected tissues, as well as the results of the adsorption and internalization assays. all p-values were two-tailed and differences were considered significant when p < . . in addition, kruskal-wallis anova followed dunn's multiple comparison tests in spss statistics were used to compare the trachea lesion scores and clinical scores. differences in the survival rates were analyzed using the log-rank test. we constructed seven full-length cdna clones where the hvrs from beaudette were substituted with those from ck/ch/ldl/ either separately or simultaneously (fig. a and table ). the rna transcripts generated from the full-length cdna clones were introduced separately into vero cells together with the n transcripts by electroporation (fang et al., ) . after h, the virus present in each medium was collected and used to inoculate -day-old spf embryonated eggs. to differentiate the rescued viruses from the parental viruses, viral rna was extracted from allantoic fluid and the s genes were amplified using rt-pcr, where the rt-pcr products covering the three hvrs were sequenced. viral antigen was observed in vero cells infected with the beaudette strain and the seven recombinant ibvs (fig. b) . no virus antigen was found in cells infected with ck/ch/ldl/ . rt-pcr analysis of subgenomic mrna and mrna was performed to confirm the virus replication (fig. c) (fang et al., ) . the results confirmed that the recombinant ibvs were adapted to vero cells in a similar manner to the beaudette strain. to test whether exchanging the hvrs affected the growth properties and genetic stability of the rescued viruses, the recombinant ibvs were propagated on embryonated eggs or vero cells for three passages. the vero cells were cultured in -well plates and infected with the recombinant ibvs and two parental strains ( × eid ) for h at °c or h at °c. the experiments were conducted based on three replicates. (a) infected cells were fixed and celisa was performed as described in the materials and methods. results are shown as viral load adsorption on cell equivalents relative to eid compared with that in the beaudette strain ( %). (b) vero cells were cultured in -well plates and infected with the recombinant ibvs and parental strains ( × eid ) for h at °c. total rna was extracted from the infected cells before quantifying the viral loads using real-time rt-pcr. data were normalized against the gapdh expression levels and viral rna was calculated relative to that in the beaudette strain ( %). the experiments were conducted based on three replicates. (c) celisa was performed to evaluate the viral load internalized into cells. (d) real-time rt-pcr was also used to evaluate the viral load internalized into cells. the experiments were conducted based on three replicates. differences were considered significant at p < . using anova followed tukey's multiple comparison tests. s gene sequencing results confirmed that the heterogenous hvrs were stably maintained in the recombinant ibvs (sup fig. ) , and no additional mutations were detected in the s protein after three passages in cells or eggs. the growth properties of the recombinant ibvs were then determined in vero cells and embryonated eggs. in vero cells (fig. c) , the growth kinetics of the recombinant ibvs were similar to those of the beaudette strain, and they all reached their peak titer at h post-infection, and there was no significant difference between the titers for the recombinant ibvs and beaudette at different time points (fig. ) . in embryonated eggs (fig. a) , although there were no significant difference between the virus titers for the recombinant ibvs and beaudette at different time points, the titers of ck/ch/ldl/ was significantly higher than those of the recombinant ibvs and beaudette at , , , h (fig. b ). in conclusion, the recombinant ibvs exhibited similar growth phenotypes compared to the beaudette strain in both cells and eggs. the if staining results showed the ibv n antigens in vero cells infected with the recombinant ibvs (fig. b) and they have the similar ability to replicate in vero cells with ibv beaudette. we then evaluated the effects of the heterogenous hvrs on virus adsorption and internalization. the celisa results for the adsorption assay showed that compared with beaudette, the recombinant ibvs exhibited significantly reduced adsorption at °c for h (fig. a) . the real-time rt-pcr results were consistent with the celisa results in the adsorption assays (fig. b) . the results indicated that the heterogenous hvrs could reduce the adsorption capacity of the recombinant ibvs. in addition, the celisa results from the internalization assay showed that the viral yields of the recombinant ibvs internalized into cells did not differ significantly from those with beaudette (fig. c) . the real-time rt-pcr results were consistent with the celisa results from the internalization assay (fig. d) . in order to test the pathogenicity of the recombinant ibvs, -day-old spf chickens were inoculated via intranasal and ocular routes at a dose of × eid per bird (sun et al., ) . we found that some of the chickens only had very low levels of snicking at and days postinfection, and there were no obvious clinical signs in the groups treated with the recombinant ibvs and beaudette. however, / birds ( %) died during - days post-challenge with ck/ch/ldl/ and the morbidity rate was % (fig. a) . the mean scores for the clinical signs in the birds inoculated with the recombinant ibvs and beaudette were much lower than those treated with ck/ch/ldl/ (fig. b) . the autopsy results showed that ck/ch/ ldl/ caused obvious swollen pale kidneys, where the tubules and ureters were distended with urates, thereby indicating the nephropathogenic potential of the virus. however, the autopsies detected almost no changes in the groups treated with the recombinant ibvs and beaudette. the h&e staining results also showed that there were no obvious pathological change in the lungs, cecum tonsils, and kidneys in the groups treated with the recombinant ibvs and beaudette (sup fig. ) . we also assessed the severity scores for histopathological lesions in the trachea with deciliation (fig. a) , goblet cells (fig. b) , mucosal thickness (fig. c) , and lymphocytes (fig. d) at days after challenge with the ibvs (van ginkel et al., ) . the scores were lowest in the beaudette group and highest in the ck/ch/ ldl/ group, and there were no differences in the scores between the groups treated with the recombinant ibvs and beaudette. however, the scores in the groups treated with the recombinant ibvs and beaudette differed significantly from those with ck/ch/ ldl/ . finally, no ibv rna was detected in the oropharyngeal swabs from chickens inoculated with the recombinant ibvs and beaudette after , , , , and days by real-time rt-pcr, where the results were considered positive when the ct value was less than zhao et al., ) . the results confirmed that the recombinant ibvs were avirulent according to the histomorphometry and histopathology findings. using a previously described method (archetti and horsfall et al., ) , the serotype relatedness values were calculated based on the cross-virus neutralization studies using vero cells (table ) or embryonated chicken eggs (table ) . viruses with an archetti and horsfall relatedness value greater than were considered to be related serotypes. our results showed that the recombinant ibvs and beaudette belonged to the same serotype (table ) , whereas the recombinant ibvs and ck/ch/ldl/ belonged to different serotypes (table ). in general, we found that the serotype relatedness values decreased between the recombinant strain and beaudette (table ) as the number of substituted heterogenous hvrs increased. these results confirmed that the replacement of the heterogenous hvrs had not caused serotype switches in this case. the ihc results showed that the viral antigen of ck/ch/ldl/ was detected in trachea (sup fig. a) , cecal tonsil (sup fig. b) , and kidney samples (sup fig. c ), but the results were negative in the tissues infected with the recombinant ibvs and beaudette. the results were also negative in all of the lung samples (sup fig. d) . the viral rna could be detected by real-time rt-pcr in the selected tissues from chickens infected with ck/ch/ldl/ , with high viral loads in the kidneys. however, very low rna levels were detected in only a few (a) mortality was recorded daily for randomly selected infected birds and the survival curve was drawn. (b) at days after challenge, we evaluated the challenged chickens blindly and clinical signs were scored as: = absent, = mild, = moderate, or = severe. kruskal-wallis anova followed dunn's multiple comparison tests were employed to perform comparisons of the clinical scores using spss. d. shan et al. virus research ( ) - trachea samples from the birds infected with the recombinant ibvs and beaudette (fig. ) . these results confirmed that the recombinant ibvs had the same tissue tropism as the beaudette strain. inoculating chickens with the seven recombinant ibvs induced clinical protection against challenge with ck/ch/ldl/ (table ). viral shedding was detected in some of the chickens challenged with ck/ch/ldl/ , but inoculating chickens with the seven recombinant ibvs and beaudette reduced the morbidity and mortality rates compared with birds in the pbs group. however, the morbidity and mortality of the recombinant ibvs and beaudette vaccinated groups was not as low as that of ck/ch/ldl/ inoculated group. these results suggest that although the recombinant ibvs could fig. . trachea lesion scores. at days after challenge, the other five infected birds in each group were killed humanely using carbon dioxide over inhalation. tissue samples were collected from the trachea and analyzed by h&e staining. mucosal thickness (a), deciliation (b), goblet cells (c) and lymphocyte scores (d) for the trachea samples were evaluated blindly and scored from to based on severity. kruskal-wallis and dunn-bonferroni tests were employed to perform post hoc comparisons of trachea lesion scores using spss. virus replication was found in vero cells infected with beaudette and the recombinant ibvs, so the neutralization tests between them were performed in vero cells. not offer complete protection against challenge with ck/ch/ldl/ , inoculation with the seven recombinant ibvs did confer some cross protection against ck/ch/ldl/ challenge. coronavirus ibv s protein has multiple biological functions during the viral replication cycle. previous studies suggest that some of these functions may be associated with hvrs in the s subunit of the s protein (cavanagh and davis, ; ignjatovic and galli, ; johnson et al., ; song et al., ) . these previous studies employed forward genetics methods to determine the effects of the hvrs on serotypes by characterizing phylogenetically closely related isolates. however, these strains inevitably had mutations in other parts of the ibv genome, thereby making it difficult to establish a precise model for further studies based on these previous findings. in the present study, we manipulated the ibv rna genomes by reverse genetics to produce site-directed mutations in order to elucidate the specific biological functions of the hvrs. we used the beaudette and ck/ch/ldl/ strains in our study. ibv beaudette is a well-known apathogenic lab strain (geilhausen et al., ) , which was adapted to vero cells from chicken embryos (fang et al., ) . the ck/ch/ldl/ ibv strain is an epidemic strain isolated from h -vaccinated layers in china. this strain mainly causes gross lesions in chicken kidneys, with high morbidity and mortality rates (liu et al., (liu et al., , liu et al., b) . the ck/ch/ldl/ strain can only proliferate in chicken embryos and not vero cells. in addition, the beaudette and ck/ch/ldl/ strains belong to different serotypes. thus, the differences in these two strains facilitate research into the effects of the hvrs in intact viral particles on serotypes, virulence, and tropisms. in order to determine the roles of individual hvrs and the accumulated effects of hvrs, we constructed seven recombinant ibvs where the three hvrs in the beaudette strain were separately or simultaneously substituted with those from ck/ch/ldl/ . all of the recombinant ibvs proliferated in vero cells (figs. b and figure ) , and had the similar growth kinetics as beaudette in vero cells (fig. ) and embryonated eggs (fig. b) . the accumulation of heterogenous hvrs weakened the capacity for adsorption during infection by the recombinant viruses in vitro ( fig. a and b) . the hvrs may be associated with the receptor-binding domain, so the heterogenous hvrs weakened the interaction between the viruses and their receptors on the host cells. with the same sequences of fusion subunit s , the recombinant ibvs and beaudette did not differ significantly in the internalization assay. it is unclear whether the recombinant viruses have more efficient internalization than beaudette and the s proteins assemble correctly in the recombinant viruses, and whether there is a comparable amount of s on the virus particle. these problems require for further investigation. both the recombinant ibvs and beaudette only weakly infected the trachea in -day-old spf chickens (fig. e) , whereas ck/ch/ldl/ infected multiple chicken tissues, including the trachea, lungs, kidneys, and cecal tonsils. thus, the replication capacity was similar for the recombinant ibvs and the beaudette strain in spf chickens. it appears that only swapping the hvrs did not greatly affect the cell and tissue tropisms, although replacing the ectodomain of the s glycoprotein in the beaudette strain can alter the growth characteristics (casais et al., ) . virus binding to host cells is the first step in tropism determination and hvrs within the s subunit may affect ibv binding (wickramasinghe et al., ) , but s is responsible for membrane fusion , and thus exchanging the hvrs did not change their tropism. fig. . detection of ibv replication in challenged chickens. at days after challenge, the other five infected birds in each group were killed humanely using carbon dioxide over inhalation, and tissue samples of the trachea, lungs, kidneys, and cecum tonsils were collected to determine the presence of ibv by real-time rt-pcr. a ct value less than was considered to be ibv-positive using real-time rt-pcr. the numbers of tissue samples positive for ibv rna/the number detected are presented at the bottom. bars in different colors represent different tissues from chickens, as indicated in the graphical representation. the average copy numbers of ibv rna in the positive samples are shown. differences were considered significant at p < . using anova followed tukey's multiple comparison tests. d d d d d d d d d d d d d d rhvr i / / / / / / / / / / / / / / / / / rhvr ii / / / / / / / / / / / / / / / / / rhvr iii / / / / / / / / / / / / / / / / / rhvr i/ii / / / / / / / / / / / / / / / / / rhvr i/iii / / / / / / / / / / / / / / / / / rhvr ii/iii / / / / / / / / / / / / / / / / / rhvr i/ii/iii / / / / / / / / / / / / / / / / / beaudette / / / / / / / / / / / / / / / / / ck/ch/ldl/ / c / / / / / / / / / / / / / / / / pbs / / / / / / / / / / / / / / / / / a viral rna was detected in oral swab samples by real-time rt-pcr and samples with a ct value less than were considered positive. b days after challenge. c five birds died days after inoculation with ck/ch/ldl/ . previous studies have shown that the s protein has major effects on pathogenicity in coronaviruses (leparc-goffart et al., ; phillips et al., ) , but the specific effects of hvrs in the s protein on pathogenicity are not clear. in the present study, the recombinant ibvs remained avirulent with the hvrs from pathogenic ck/ch/ldl/ , thereby indicating that only exchanging the hvr sequences did not have major impacts on pathogenicity (figs. and , sup. fig. ). according to hodgson et al., the apathogenic nature of the recombinant ibv beaur-m (s) indicates that the s protein ectodomain from a virulent strain is not necessarily sufficient to overcome the attenuating mutations in other genes in the apathogenic beaudette strain (hodgson et al., ) . it is possible that the replicase gene also contributes to the pathogenicity of ibv (armesto et al., ; hodgson et al., ) . the functions of non-structural proteins (nsp) in the context of pathogenesis are still not well understood, but some of the nsps in other coronaviruses have been linked to loss of pathogenicity (eriksson et al., ; sperry et al., ) . it is considered that the hvrs of the ibv s subunit may induce abundant virus neutralizing antibodies koch et al., ; moore et al., ; niesters et al., ) . however, in our study, the introduction of heterogenous hvrs did not develop an independent serotype, although the antigenic relatedness values for the recombinant ibvs and beaudette generally decreased as the number of heterogenous hvr exchanges increased (table ). this result is consistent with the conclusion of santos fernando et al. who found that three ibv isolates mainly exhibited mutations in the hvrs but they belonged to the same serotype (santos fernando et al., ) . the repertoire of neutralizing polyclonal antibodies may react with the many epitopes of an antigen. exchanging the hvrs could change several antigen determinants but it is not necessarily sufficient to change the serotype because other neutralizing epitopes have been identified in other parts of the s subunit and s subunit (ignjatovic and sapats, ; kusters et al., ; lenstra et al., ) . in particular cases, a very low number of critical amino acid changes is sufficient to greatly alter the antigenicity (cavanagh et al., ) , although this view does not apply in all cases (chen et al., ) . in conclusion, the recombinant ibvs exhibited differences in antigenicity, but exchanging only the three hvrs between the two parental strains did not change the serotype. vaccination is considered the most cost-effective approach for controlling ibv infection. however, current commercial vaccines have been challenged by the emergence of new ibv serotypes. recently, reverse genetic techniques have been employed to modify ibv vaccine candidates. qx-like strains (lx -type) emerged in china and spread to asia (mahmood et al., ) , russia (bochkov et al., ) , and europe (beato et al., ; worthington et al., ) . in this study, using ck/ ch/ldl/ as a representative of epidemic qx-like strains, we modified the genome of the beaudette strain to construct seven recombinant ibvs. we determined the effects of the hvrs and explored the possibility of developing a vaccine candidate that could proliferate in cells and provide protection against the prevalent field strains. the introduction of a few heterogenous peptides rather than the whole s protein may facilitate the development of multiepitope peptide vaccines to protect against a wide range of ibv serotypes (yang et al., ). in the present study, the virus cross-neutralization and vaccination challenge tests indicated that only swapping the hvrs did not provide sufficient cross-protection, but further exploration of the recombinant ibvs may provide insights into novel vaccine candidates. in conclusion, we manipulated the genome of the ibv beaudette strain using a reverse genetics system to construct seven recombinant strains by replacing the hvrs in the beaudette 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induced by multi-epitope based peptide vaccines serotype shift of a /b genotype infectious bronchitis coronavirus by natural recombination recombinant newcastle disease virus expressing the infectious bronchitis virus s gene protects chickens against newcastle disease virus and infectious bronchitis virus challenge this work was supported by grants from the china agriculture research systerm (no. cars- -k ), the provincial supported science foundation of heilongjiang province for the national key technology r&d program (gx b ) and national "twelfth five-year" plan for science & technology support ( bad b ). the authors declare that they have no competing interests. all authors declare that they have no conflict of interest. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.virusres. . . . key: cord- -k hlf authors: sun, dongbo; shi, hongyan; guo, donghua; chen, jianfei; shi, da; zhu, qinghe; zhang, xin; feng, li title: analysis of protein expression changes of the vero e cells infected with classic pedv strain cv by using quantitative proteomic technique date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: k hlf recent outbreaks of porcine epidemic diarrhea virus (pedv) have caused widespread concern. the identification of proteins associated with pedv infection might provide insight into pedv pathogenesis and facilitate the development of novel antiviral strategies. we analyzed the differential protein profile of pedv-infected vero e cells using mass spectrometry and an isobaric tag for relative and absolute quantification. a total of proteins were identified that were differentially expressed between the pedv-infected and mock-infected groups (p < . , quantitative ratio ≥ . ), among which the expression of proteins was up-regulated and that of proteins was down-regulated in the pedv-infected vero e cells, involving in integrin β /β , cystatin-c. the gene ontology analysis indicated that the molecular function of the differentially expressed proteins (deps) was primarily related to binding and catalytic activity, and that the biological functions in which the deps are involved included metabolism, organismal systems, cellular processes, genetic information processing, environmental information processing, and diseases. among the disease-related functions, certain anti-viral pathways and proteins, such as the rig-i-like receptor, rap , autophagy, mitogen-activated protein kinase, pi k-akt and jak-stat signaling pathways, and integrin β /β and cystatin-c proteins, represented potential factors in pedv infection. our findings provide valuable insight into pedv-vero e cell interactions. the porcine epidemic diarrhea virus (pedv) is an enveloped, single-stranded positive-sense rna virus that causes porcine epidemic diarrhea (ped), an acute and highly contagious enteric disease in pigs. ped is characterized by severe diarrhea, vomiting, dehydration, and a mortality rate of up to % in suckling piglets (pensaert and debouck, ) . ped was first reported in belgium and the united kingdom in , and frequent outbreaks have occurred in various asian countries . since , acute ped outbreaks have continually occurred in thailand, china, and the usa, which have resulted in substantial economic losses (puranaveja et al., ; li et al., ; chen et al., ; huang et al., ; marthaler et al., ; stevenson et al., ; yang et al., ; chen et al., ) . the continued outbreaks of ped, despite control efforts, have caused widespread concern. the pedv belongs to the genus alphacoronavirus, in the family coronaviridae and order nidovirales (belouzard et al., ) . previous studies have investigated various control measures to protect against pedv infection, such as vaccines, diagnostic tools, and therapeutic drugs (sun et al., ; ren et al., ; sun et al., ; zhu et al., ; guo et al., ; kim and lee, ) . various aspects of pedv infection remain unclear, for example, swine testis (st) cells expressing porcine aminopeptidase n of pedv receptor were not susceptible to pedv infection. african green monkey kidney (vero) cells are highly susceptible to pedv infection, and are widely used for the primary isolation and cultivation of pedv guo et al., ) . therefore, vero lineages are suitable hosts for understanding the mechanisms of pedv infection. proteomics techniques are effective tools for characterizing protein expression profiles, and have been used widely to investigate disease-associated proteins (hondermarck et al., ; boja et al., ; he et al., ; sun et al., ) . among current proteomics methods, quantitative high-throughput proteomics approaches are useful for the analysis of infection-associated proteins of pathogens (linde et al., ; papachristou et al., ; ye et al., ; zeng et al., ) . in our current study, we used a quantitative proteomics approach based on an itraq tandem mass spectrometry (ms/ms) technique to identify proteins differentially expressed between pedv-infected and mock-infected vero e cells. the functions of the differentially expressed proteins (deps) were analyzed to determine whether they might be associated with pedv infection. our findings provide valuable insight into the changes in cellular processes that occur during pedv infection. the cv strain of pedv, kindly provided by maurice pensaert at ghent university (merelbeke, belgium), was used in all of our experiments after being adapted to vero e cells, as previously described (hofmann and wyler, ) . the vero e cell-adapted pedv, the vero e cells, and the monoclonal antibody against the nucleocapsid protein (np) of pedv were stored at the diarrhea-related viruses section, division of swine infectious diseases, national key laboratory of veterinary biotechnology, harbin veterinary research institute of the chinese academy of agricultural sciences. the vero e cells were cultured in dulbecco's modified eagle's medium containing % fetal bovine serum (fbs) in -cm flasks at • c in a % co atmosphere. when the cells reached - % confluence, they were inoculated with the pedv at a multiplicity of infection of in presence of g/ml trypsin. at h postinoculation, the cells began to exhibit cytopathic effects (cpes) of viral infection, but no cells lysis or shedding had occurred. the cells were washed three times with cold phosphate-buffered saline (pbs, ph . ). a . -ml aliquot of lysis buffer containing % sds, mm dtt, and mm tris-hcl (ph . ) was added to each flask, and the flasks were incubated at • c for min. the cell lysates were collected using a cell scraper, and boiled for min. three cell lysate replicates were prepared for the pedv-infected (v -v ) and mock-infected (c -c ) vero e cells, and stored at − • c. western blotting was performed to confirm pedv infection by detecting the presence of the np of pedv in the vero e cells. aliquots of the cell lysates were subjected to sds-page on a % acrylamide gel, and the protein bands were transferred to a nitrocellulose membrane using a semi-dry transfer device (bio-rad, hercules, ca, usa). the membrane was blocked using % (w/v) nonfat dried milk in pbs at • c for h, before incubation in pbs containing the anti-np monoclonal antibody ( : dilution) at • c for h. after washing three times with % tween in pbs (pbst), the membrane was incubated in pbst containing a horseradish peroxidase-conjugated goat anti-mouse igg ( : dilution) at • c for h. after washing three times with pbs, the membrane was incubated with enhanced chemiluminescence detection reagents (biotopped, beijing, china) at room temperature for min, and the peroxidase-mediated luminescence was digitally captured using the molecular imager chemidoc xrs+ system (bio-rad) and the image lab software (bio-rad). to verify the differential expression of the selected deps, equivalent volumes of the cell lysate replicates from the pedv-infected (v -v ) and mock-infected (c -c ) vero e cells were pooled into the v and c samples, respectively, and western blotting was performed as described above, with the following exceptions: a : dilution of the polyclonal antibodies anti-␤ tubulin, anti-integrin-␤ , anti-cystatin-c, anti-protein s -a , anti-apolipoprotein e , and anti-centrin from rabbit (beijing biosynthesis biotechnology, beijing, china) was used as the primary antibody, and a : dilution of the hrp-conjugated goat anti-rabbit igg (sigma-aldrich, st. louis, usa) was used as the secondary antibody. protein digestion of the samples was performed according to the fasp procedure described by wiśniewski et al. ( ) . an aliquot of each cell lysate containing g of protein was combined with l of std buffer containing % sds, mm dtt, and mm tris-hcl (ph . ). the detergent, dtt, and other low-molecular-weight components were removed by dilution in ua buffer containing m urea and mm tris-hcl (ph . ) and repeated ultrafiltration using microcon ( kda) ultrafiltration units. the reduction of cysteine residues was blocked by the addition of l of . m iodoacetamide to the ua buffer. the samples were incubated for min in darkness before ultrafiltration. the microcon filters were washed three times with l of ua buffer, followed by two washes with l ds buffer containing mm triethyl ammonium bicarbonate (ph . ). the final protein suspensions were digested using g of trypsin (promega, madison, wi, usa) in l of ds buffer overnight at • c, and the digested peptides were collected as the filtrate. the peptide content was quantified based on absorbance at nm using an extinction coefficient of . for a . mg/ml solution. the digested peptide mixture was labeled using the -plex itraq reagent (life technologies, carlsbad, ca, usa), according to the manufacturer's instructions. each itraq reagent was dissolved in l of ethanol, and added to the digested peptide mixture. the samples were labeled as c - , c - , c - , v - , v - , or v - . the samples were multiplexed, and vacuum dried. the itraq labeled peptides were fractionated by scxc using the akta purifier system (ge healthcare, waukesha, wi, usa). the dried peptide mixture was reconstituted, and acidified by the addition of ml of buffer a containing mm kh po in % acetonitrile (ph . ). the samples were loaded onto a . mm × mm column packed with polysulfoethyl ( m, Å) chromatography resin (polylc, columbia, maryland, usa). the peptides were eluted at a flow rate of ml/min using a gradient of - % buffer b containing mm kcl and mm kh po in % acetonitrile (ph . ). the gradient elution consisted of - % buffer b for min, - % buffer b for min, and - % buffer b for min. the absorbance of the eluate was monitored at nm, and fractions were collected at -min intervals. thirty fractions were combined into ten pools, and desalted using empore standard density spe c cartridges (sigma-aldrich, st. louis, mo, usa) with a bed diameter of mm and a volume ml. each fraction was concentrated by centrifugation in a vacuum, and reconstituted in l of . % (v/v) trifluoroacetic acid. all samples were stored at − • c until the ms analysis was performed. the lc-ms/ms experiments were performed using a q exactive mass spectrometer coupled to a proxeon biosystem easy nanolc (thermo fisher scientific, waltham, ma, usa). ten microliters of each fraction was injected for nanolc-ms/ms analysis. the peptide mixture ( g) was loaded onto a c -reversed phase column ( cm × m) packed with rp-c ( m) resin in buffer a containing . % formic acid, and eluted with a linear gradient of buffer b ( % acetonitrile and . % formic acid) at a flow rate of . l/min for min using the intelliflow technology. the eluate underwent electrospray ionization for the ms/ms analysis. the ms/ms instrument was run in the peptide recognition mode, and the spectra were acquired using a data-dependent top- method based on the selection of the most abundant precursor ions from the survey scan ( - m/z) for hcd fragmentation. the determination of the target value was based on the predictive automatic gain control, and the dynamic exclusion duration was s. survey scans were acquired at a resolution of , at m/z , and the resolution for the hcd spectra was set to , at m/z . the normalized collision energy was ev, and the underfill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as . %. the ms/ms spectra were compared to the uniprot cercopithecidae database ( sequences, downloaded november , ) and a decoy database using the mascot search engine, version . (matrix science, london, uk), embedded in the proteome discoverer . software (thermo electron, san jose, ca). the following parameters were used for protein identification: a peptide mass tolerance of ppm; an ms/ms tolerance of . da; trypsin digestion; a missed cleavage value of ; the fixed modifications included carbamidomethyl, itraq plex(k), and itraq plex(n-term); the variable modification was oxidation; and an fdr value ≤ . . protein quantification was performed using the proteome discoverer . software based on the centroided reporter ion peak intensity. the average quantitative value of each protein in samples c , c , and c (mock-infection group) was used as the internal reference. the value of the quantitative ratio for each protein relative to the internal reference was calculated, and averaged to obtain the quantitative ratio (v/c) of the proteins identified in the treatment groups (unwin et al., ) . a protein was considered to be differentially expressed between the pedv-infected and mock-infected groups based on the following criteria: the protein had to be present in three replicates of both groups, the difference in the level of the protein between the two groups had to be statistically significant (p < . ), and the change ratio for the protein had to be ≥ . (yuan et al., ) . the expression of a protein with a v/c > . was considered to be up-regulated, and those with a v/c < . were considered to be down-regulated. the data were analyzed using a two-tailed, paired student's t test. the statistical analysis was performed using the excel software (microsoft, redmond, wa, usa). the deps were annotated using the blast go, version . . , program (ashburner et al., ; quevillon et al., ; götz et al., ) . the deps were blasted against the kegg genes database (human). the gene ontology categories (gocs) were retrieved, and mapped to pathways in the kegg database (kanehisa et al., ) . table the proteins identified from pedv-infected and mock-infected groups. the vero e cells inoculated with pedv displayed distinct cpes at h postinoculation, including cell shrinkage, cell fusion, and a rounded cell morphology, but no cells lysis or shedding was observed (fig. a) . the immunoblotting analysis confirmed that the vero e cells were pedv-infected. the band corresponding to the np of pedv was detected in samples v , v , and v , whereas none was detected in samples c , c , and c (fig. b) . the identified peptides, identified proteins, quantified proteins, known/uncharacterized proteins, and the goc annotations are showed in table . a total of proteins, including peptides, were identified in the pedv-infected and mock-infected groups using the itraq-ms/ms approach, among which ( . %) were quantified, ( . %) were known proteins, and ( . %) were uncharacterized/putative proteins. based on the gocs, ( . %) of the proteins were annotated as biological process, ( . %) were annotated as molecular function, and ( . %) were annotated as cellular components. the quantification and significance of the identified proteins are shown in fig. . the changes in the levels of expression between the two groups were analyzed based on statistical significance. of the proteins identified, ( . %) were not differentially expressed (p > . ), and ( . %) were expressed at statistically different levels between the pedv-infected and mockinfected vero e cells (p < . ), including proteins ( . %) with a p-value between . and . , proteins ( . %) with a p-value between . and . , and proteins ( . %) with a p-value < . . the proteins with a p-value < . were also filtered based on whether the v/c or c/v was ≥ . . based on these criteria, a total of ( . %) of the identified proteins were determined to have been differentially expressed between the pedv-infected and mock-infected groups (table ) . among the deps, . % ( / ) were up-regulated, and . % ( / ) down-regulated. the known proteins and uncharacterized/putative proteins accounted for . % ( / ) and . % ( / ) of the deps, respectively. the dep displaying the greatest increase in expression in the pedv-infected vero e cells was isoform of the ovarian cancer immunoreactive antigen domaincontaining protein ( : . ), and the dep displaying the greatest decrease in expression in the pedv-infected vero e cells was cystatin-c ( : . ). the gene ontology (go) database has been widely used for describing protein function in a standardized format. according to their gocs, the deps were annotated as cellular component, biological process, or molecular function. the go annotations are shown in table , and distributions of the go annotations are shown in fig. . seventy-eight deps were distributed among groups of biological processes (fig. a) . the metabolic process (go: ), cellular process (go: ), single-organism process (go: ), and biological regulation (go: ) groups contained the highest proportions of the biological process deps. there were more up-regulated proteins in the cellular component organization group (go: ) than down-regulated proteins. seventy-four deps were distributed among eight cellular component groups (fig. b) , among which the organelle (go: ) and cell (go: ) groups contained the highest proportion of cellular component deps. there were more down-regulated deps in the membrane group (go: ) than up-regulated deps, and there were more up-regulated deps in the macromolecular complex group (go: ) than downregulated deps. ninety-seven deps were distributed among eight molecular function groups (fig. c) , among which the binding (go: ) and catalytic activity (go: ) groups contained the greatest proportion of molecular function deps. the kyoto encyclopedia of genes and genomes (kegg) pathway is a collection of pathway maps that represent molecular interactions and reaction networks in cells. seventy-five of the deps identified were annotated, and mapped to a total of six kegg pathway categories, which included the metabolism, organismal systems, cellular processes, genetic information processing, environmental information processing, and diseases pathway categories (fig. ) . the annotations in the metabolism, organismal systems, and diseases pathway categories represented , , and pathway groups, respectively (fig. a, b and f). the annotations in metabolism pathways category included the carbohydrate, energy, lipid, nucleotide, amino acid, glycan biosynthesis, cofactors and vitamins, biosynthesis of other secondary metabolites, and xenobiotics pathway groups (fig. a ). the annotations in the organismal systems category included the tolllike receptor (tlr) signaling (ko ), rig-i-like receptor (rlr) signaling (ko ), and natural killer cell mediated cytotoxicity (ko ) pathway groups (fig. b) , which represent pathways related primarily to the immune response to virus infection. the largest number of deps in the cellular process category were mapped to the lysosome (ko ) pathway group, all ten of which were down-regulated deps (fig. c ). the annotations in the genetic information processing category included pathway groups related to dna replication and repair, transcription, translation, and the folding, sorting, and degradation of proteins (fig. d ). the annotations in the environmental information processing proteins and one up-regulated protein. overall, more disease pathway groups were assigned to a single down-regulated dep than those assigned to up-regulated deps. the integrin (␤ and ␤ subunits) protein was annotated to the largest number of pathway groups ( ), which included the organismal systems, environmental information processing, cellular processes, and diseases categories. the ␤ tubulin as loading control, three down-regulated deps cystatin-c, apolipoprotein e and centrin- , two up-regulated deps integrin-␤ and protein s -a , were selected to verify differential expression between the pedv-infected and mock-infected vero e cells. the immunoblotting analysis showed that the ratios of these proteins between the pedv-infected and mock-infected groups were consistent with those obtained using the quantitative proteomics analysis (fig. ) . in our study, pedv infection significantly alters protein expression in vero e cells. the differentially expressed proteins (deps) annotated to virus infection-associated signaling pathways, autophagy, and virus entry-associated proteins were analyzed further to assess their potential roles in pedv infection. in mammals, the first line of defense against virus infection is the innate immune system. early antiviral responses are initiated upon the recognition of pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs), resulting in the production of interferons for the innate immune response and the maturation of dendritic cells for establishing acquired immunity (yokota et al., ) . the prrs are grouped into the tlrs, rlrs, and nucleotide binding-oligomerization domain-like receptors. our results showed that pedv infection induced the deps that participated in six signaling pathways involved in viral infection, including the rlr, rap , pi k-akt, mapk, jak-stat, and tlr signaling pathways. the pedv is an enteric virus that infects the intestinal epithelial cells (iec) of swine, causing severe diarrhea. hirata et al. ( ) reported the rig-i signaling pathway plays an important role in antiviral innate immunity mechanisms in iecs. sheikh et al. ( ) reported the rap a signaling pathway was associated with secretory diarrhea. the jak-stat signaling pathway regulates the adaptive and innate mechanisms related to mucosal immunity (heneghan et al., ; wang et al., ) . our results showed that deps induced by pedv infection in vero e cells involved in the rlr, rap , and jak-stat signaling pathways. it has been reported that the tlr, mapk, and pi k-akt signaling pathways play roles in host cell responses to coronaviruses (mizutani et al., ; integrins are cell surface ␣/␤ heterodimeric glycoproteins that contribute to a variety of cellular functions (stewart and nemerow, ) . combinations of the various isotypes of the ␣ and ␤ subunits of integrins generate more than different integrin proteins. previous studies have shown that various integrin molecules are used as receptors for virus attachment (stewart and nemerow, ; sun et al., ) . in our current study, the expression of integrin-␤ and -␤ was down-regulated and up-regulated, respectively, in response to pedv infection. the upregulation of integrin-␤ expression is consistent with that observed in response to dengue virus infection (zhang et al., ) . our pathway analysis revealed that both integrin-␤ and -␤ are involved in pathways that contribute to organismal systems, environmental information processing, cellular processes, and diseases. the integrin ␣v␤ protein has been shown to serve as an entry receptor for various viruses (guerrero et al., ; neff et al., ; chu and ng, ; parry et al., ; wang et al., ) , some of which bind the integrin through an rgd sequence in a viral structural protein to initiate infection (stewart and nemerow, ) . the s protein of pedv is a glycoprotein peplomer on the viral surface that plays an important role in receptor-mediated binding and cell membrane fusion. in our study, the integrin recognized sequences of pedv s protein was analyzed based on ruoslahti's ( ) report. the results indicated that four conserved integrin-recognized amino acid motifs (asp-gly-glu, lys-gly-glu, arg-leu-asp, and leu-asp-val) were found in the s proteins of various pedv strains (data not shown). these data suggest that integrin proteins may act as an infection associated protein for the attachment and entry of pedv. autophagy is an essential component of host defenses against viral infection (dong and levine, ) . maier and britton ( ) reported that ␤-coronaviruses induced autophagy. in our study, more deps were mapped to the autophagy pathway group than any of the other pathway groups. fifteen deps were mapped to the lysosome and phagosome pathways. of the proteins, ( %) were down-regulated deps. although the autophagy pathway plays an antiviral role in virus-infected cells, the autophagy machinery is exploited by certain viruses for viral evasion and propagation. our results showed that pedv infection induced the downregulation of the expression of many autophagy-associated proteins. therefore, pedv infection might inhibit autophagy in vero e cells, thus facilitating virus replication. previous studies have shown that the microtubule-associated protein b is a useful biomarker protein for autophagy (dong and levine, ) . we found that the expression of map b was up-regulated . -fold in the pedv-infected vero e cells. these results suggest that the pedv induces autophagy. cystatin-c has been shown to reduce the replication of certain viruses, including the poliovirus, rhinovirus, and human coronaviruses oc and e (korant et al., ; collins and grubb, ) . the cleavage of s protein has been shown to be essential for the induction of cell-to-cell fusion and coronavirus entry into cells (sturman et al., ) . shirato et al. ( ) reported the transmembrane type ii serine protease enhanced infection of pedv in vero cells by increasing virus release. in our study, the reduced expression of cystatin-c might facilitate pedv replication and release through the activation of cysteine-associated proteases in vero e cells. apolipoprotein e , galectin, clusterin, and transferrin receptor have also been shown to be associated with virus infection (hishiki et al., ; peng et al., ; martin and uprichard, ; tripathi et al., ) , and may therefore function as infectionassociated proteins in pedv-infected vero e cells. additionally, the decreased in vitro expression of the adherens junction protein, such as cadherin, might be associated with a reduced integrity of pedv-infected intestinal epithelial cells in vivo. to the best of our knowledge, our study represents the analysis of the interactions between pedv and vero e cells using a quantitative proteomics technique. pedv infection-associated pathways and proteins are described and discussed based on the bioinformatics analysis of the differentially expressed proteins. our analysis of vero e cell responses to pedv infection identified relevant targets for subsequent in-depth studies of pedv pathogenesis, expand the current knowledge base regarding the interaction between the pedv and the host cell, and provide useful basic information about other coronaviruses. although the vero e cells are highly susceptible to pedv infection and facilitate experimental design and performance for proteomics, the vero e cell line is an interferondeficient cell line and not a pig cell line. so, the detailed functions of these pathways and proteins in pedv infection require further verification in the actual host cells of pedv. gene ontology: tool for the unification of biology mechanisms of coronavirus cell entry mediated by the viral spike protein evolution of clinical proteomics and its role in medicine detection and molecular diversity of spike gene of porcine 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single-walled carbon nanotubes and graphene oxide on human hepatoma hepg cells: an itraq-coupled d lc-ms/ms proteome analysis proteome analysis of porcine epidemic diarrhea virus (pedv)-infected vero cells up-regulated expression of beta integrin induced by dengue virus serotype infection associated with virus entry into human dermal microvascular endothelial cells expression and purification of the scfv from hybridoma cells secreting a monoclonal antibody against s protein of pedv this work is supported by the national natural science foundation of china (grant no. ), the state national key laboratory of veterinary biotechnology (grant no. sklvbf / ), and the program for new century excellent talents in heilongjiang provincial university (grant no. -ncet- ). key: cord- -et sli authors: du, ruikun; wang, lili; xu, hao; wang, zhiying; zhang, tao; wang, manli; ning, yunjia; deng, fei; hu, zhihong; wang, hualin; li, yi title: a novel glycoprotein d-specific monoclonal antibody neutralizes herpes simplex virus date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: et sli the worldwide prevalence of herpes simplex virus (hsv) and the shortage of efficient vaccines and novel therapeutic strategies against hsv are widely global concerns. the abundance on the virion and the major stimulus for the virus-neutralizing antibodies makes gd a predominant candidate for cure of hsv infection. in this study, we generated a monoclonal antibody (mab), termed m f, targeting to glycoprotein d (gd) of hsv- , which also has cross-reactivity against hsv- gd. it has a high level of neutralizing activity against both hsv- and hsv- , and binds to a highly conserved region (residues – ) within the pro-fusion domain of gd. it can effectively block hsv cell-to-cell spread in vitro. the pre- or post-attachment neutralization assay and syncytium formation inhibition assay revealed that m f neutralizes hsv at the post-binding stage. moreover, therapeutic administration of m f completely prevented infection-related mortality of mice challenged with a lethal dose of hsv- . our newly identified epitope for the neutralizing antibody would facilitate studies of gd-based hsv entry or vaccine design, and m f itself demonstrated a high potential for adaptation as a protective or therapeutic drug against hsv. herpes simplex virus (hsv) is a prevalent worldwide human pathogen that infects epithelial cells before it establishes latency in trigeminal or sacral nerve root ganglia (steiner and benninger, ) , causing mucocutaneous lesions, keratitis, and encephalitis (dropulic and cohen, ) . there are two serotypes of hsv, hsv- and hsv- , also known as human herpesvirus and (hhv- and hhv- ). the most common clinical sign of hsv- is herpes labialis, as well as ocular diseases, including conjunctivitis and keratitis (everett, ) . hsv- is the main cause of recurrent genital herpes, which is one of the most predominant sexually transmitted diseases, and hsv- infection of newborns during delivery is usually accompanied by a high mortality rate (cleary et al., ; domeika et al., ; overall, ) . moreover, epidemiological and biological studies have revealed that hsv- dramatically increases the risk of hiv infection (barnabas and celum, ; freeman et al., ; sartori et al., ) and may also act in conjunction with human papillomavirus (hpv) infection to increase the risk of invasive cervical carcinoma (smith et al., ; zhao et al., ) . according to world health organization (who) statistics, in , there were million people aged - years living with hsv- infection worldwide (looker et al., ) , and the newly infected population is estimated to be million per year (looker et al., ) . therefore, the development of vaccines and novel therapeutic strategies against hsv has become very urgent. both initial entry of the virion into cells and subsequent lateral spread of hsv require the interaction of four viral glycoproteins, gb, gd, gh and gl, with at least one of the cell receptors, either herpesvirus entry mediator (hvem) or nectin- . among these viral glycoproteins, gd acts as the receptor-binding glycoprotein, and gh, gl and gb are required for membrane fusion execution. the gd ectodomain is organized into two structurally and functionally differentiated regions. the n terminus (residues - ) carries the receptor binding sites, and the c terminus functions as the pro-fusion domain (residues - ), which is required for triggering virus-cell fusion. in nature, the c terminus of the gd ectodomain binds to its n-terminal region and masks the receptor-binding site, resulting in a closed conformation (cocchi et al., ; fusco et al., ) . after binding to a cell surface receptor, either hvem or nectin- , gd undergoes conformational changes to adopt an open conformation, which is a key step in activating the core fusion machinery of gh/gl and gb. gd is the most abundantly expressed glycoprotein on the virion, and it induces both humoral and cellular immunity and is the major stimulus of virus-neutralizing antibodies together with gb (bender et al., ; eisenberg et al., ; fotouhi et al., ; nicola et al., ; whitbeck et al., ) . therefore, gd has been the predominant candidate for hsv vaccine or novel therapeutic strategies (awasthi and friedman, ; shin and iwasaki, ) . monoclonal antibodies (mabs) represent a useful tool for the study of the structure and function of target proteins, and virus-host interactions, as well as for the development of promising therapeutic agents. anti-gd mabs have been arranged into groups that recognize distinct discontinuous native and continuous denatured epitopes. most mabs that have virus-neutralizing activity recognize discontinuous epitopes. residues to , to , and to of the extracellular domain are major continuous antigenic determinants on gd (isola et al., ) . mabs recognizing epitopes located in these continuous determinant regions have no or low virus-neutralizing ability, except for the mab d , which can neutralize viral infectivity by blocking gdreceptor interaction (cairns et al., ) . our current investigation found a mab, m f that recognizes a new continuous epitope (residues to ) within the pro-fusion domain of hsv and possesses a high level of virus-neutralizing activity. it showed a high degree of neutralizing activity against both hsv- and hsv- , completely abrogated viral cell-to-cell spread, and inhibited syncytium formation in vitro. in addition, it also exhibited highly therapeutic effects in a hsv- infected mouse model, implying its high potential for adaptation as a protective or therapeutic interventions. cell lines used in the study are african green monkey kidney (vero) cell line (atcc, and baby hamster kidney (bhk) cell line (atcc, . cells were cultured in dulbecco's modified eagle medium (dmem) (thermo fisher scientific) with % fetal bovine serum (fbs) (thermo fisher scientific). hsv- f strain and hsv- strain were kindly provided by microorganisms and viruses culture collection center, wuhan institute of virology, cas (serial number: ivcas . , ivcas . ). viruses were propagated in vero cells and cell lysate stocks were prepared as previously described (morrison and knipe, ) . virus titers were determined in vero cells (navarro et al., ) . female balb/c mice, six-weeks of age, were purchased from the hubei provincial center for disease control and prevention (wuhan, china) and maintained under specific pathogen-free conditions at the the structure of full-length gd protein is shown at the top; the truncated gd proteins used for the sequential mapping experiment are shown below. numbers indicate amino acid positions. the domain (pro-fusion) was defined according to fusco et al. ( ) . (b) recognition region mapping of mabs m f and c . the truncated gd proteins expressed by pmal-c x vector were presented as antigens. m f and c were used as the primary antibodies respectively for western blot analysis. central animal laboratory of wuhan institute of virology, chinese academy of sciences (wiv, cas. license number: syxk - ). all animal experiments were approved by the institutional animal ethical committee of wiv, cas (approval: wiva ). all procedures were carried out under pentobarbital sodium (sigma) anesthesia and all efforts were made to minimize any suffering and the number of animals used in the study. mabs against hsv- gd protein were prepared as described previously (evan et al., ) . in brief, the nucleotide sequence encoding the ectodomain (residues to ) plus the natural signal sequence of hsv- gd protein, gd - t, was cloned into the pet- a vector (novagen) and expressed in escherichia coli bl . the protein was purified under denaturing conditions in the presence of m urea by using a ninitrilotriacetic acid (nta) column and then dialyzed (pirestani et al., ) . the quantity of purified protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). balb/c mice (n = ) were immunized with μg of purified gd - t in complete freund's adjuvant (sigma). after , , and weeks, the immunizations was repeated with gd - t conjugate in incomplete freund's adjuvant (sigma). fusion with sp / myeloma cells was carried out by using standard methodology (de stgroth and scheidegger, ) . hybridomas culture supernatant was screened by enzyme-linked immunosorbent assay (elisa) and western blot analysis. totally strains of positive hybridoma cells were obtained. the mabs were purified by protein a-sepharose™ (sigma) according to the manufacturer's instructions. for epitope mapping, the ectodomain of hsv- gd protein was dissected into seven truncated fragments, gd - t, gd - t, gd - t, gd - t, gd - t, gd - t and gd - t, with any two adjacent fragments sharing an overlap of - amino acids (fig. a) . amino acids to of the gd protein were further truncated into eight fragments, gd - t, gd - t, gd - t, gd - t, gd - t, gd - t, gd - t and gd - t (fig. a ). the nucleotides encoding the above fragments were pcramplified using the primer pairs listed in table s , cloned into pmal-c x (neb) and then expressed in e. coli dh β. for characterization of recognizing speciality, vero cells infected with hsv- or hsv- at an multiplicity of infection (moi) of pfu per cell were harvested at h post infection (p.i.) and rinsed with pbs. the protein samples were separated by % sds-page, and transferred onto a polyvinylidene fluoride (pvdf) membrane (merck millipore, massachusetts, usa) by a trans-blot apparatus (bio-rad). the western blot analyses were performed with primary antibodies generated from above-mentioned screening positive hybridoma cells which against hsv- gd protein: m f and c . pre-immune mouse serum and β-actin (sigma) antibodies were used as controls. after washing, the blots were incubated with horseradish peroxidase-conjugated goat anti-mouse antibody. the immunoreactive signals were visualized with supersignal west pico chemiluminescent substrate (fermentas) and the microchemi bioimaging system (dnr, usa). vero monolayers were infected with hsv- or hsv- at an moi of pfu per cell. at h p.i., the cells were fixed with % paraformaldehyde for min, permeabilized with . % triton x- , and washed three times with phosphate-buffered saline (pbs). to characterize the recognizing speciality of m f and c , we immunostained cells with them respectively for h at °c. pre-immune mouse serum and mouse immunoglobulin g (beyotime) were used as controls. all the antibodies were diluted : in blocking solution ( % bsa in pbs). after being washed for three times with pbs, the cells were incubated with the secondary antibody, fluorescein isothiocyanate (fitc)-conjugated goat anti-mouse igg (abcam, hangzhou, china). the cells were observed by fluorescence microscopy. the hsv neutralization assay was based on a previously described protocol (bag et al., ) . in brief, vero cell were seeded into well plates (costar) and incubated overnight to achieve confluent monolayers. m f and c were serially diluted in dmem supplemented with % fbs and then combined with hsv- or hsv- ( pfu/well) respectively (final concentration of m f or c for hsv- ranged from μg/ml to , and final concentration of m f or c for hsv- ranged from μg/ml to ). pre-immune mouse serum was used as negative control. the virus-antibody mixtures were incubated for h at °c with gentle rocking. after h incubation, the mixtures were inoculated over vero cell monolayers in plates at °c for h. after h adsorption the cells were covered with overlay media with % methylcellulose to form plaques. the plaques developed after h of incubation were fixed and visualized by staining with crystal violet. plaques were counted and the percentage inhibition was determined relative to the negative control. the effective concentration of mabs that inhibited the number of viral plaques was interpolated from the dose-response curves. virus neutralization assays were repeated at least twice and reported as number of plaques relative to control ± sem. the pre-or post-attachment neutralization assays were performed as described with modifications (krawczyk et al., ) . briefly, virusantibody mixture was incubated for h at °c before inoculating over the vero cells (pre-attachment), while prechilled ( °c for min) vero cells were infected with hsv- or hsv- ( pfu/well) at °c for h to allow virus adsorption before serial dilutions of antibodies were added (post-attachment). inoculated vero cells were then incubated for h at °c. neutralization efficacy was determined after h as described above for the standard neutralization assay. cell-to-cell spread assay was based on a previously described protocol (krawczyk et al., ) . bhk cells were grown on glass coverslips and incubated overnight to achieve confluent monolayers. cells were infected with hsv- or hsv- at an moi of . pfu per cell. after h of adsorption at °c, the virus inoculum was removed and cells were washed twice with pbs and then incubated in dmem containing % fbs in the presence of antibodies, m f ( μg/ml) and c ( μg/ml). mouse igg ( μg/ml), or medium alone was used as control. to visualize the viral spread by immunofluorescence, after h p.i., cells were fixed with % paraformaldehyde, permeabilized with . % triton x- , and washed three times with pbs. cells were incubated with mouse polyclonal antibody against gd as primary antibody and fitc-conjugated goat anti-mouse igg as secondary antibody. immunofluorescence images were observed by fluorescence microscopy. to quantify the cell-to-cell spread, the number of infected cells with gfp fluorescence per total cells was calculated in nine randomly chosen areas, representing ∼ cells for each group. the percentage infection was determined relative to control. the experiment was repeated twice. syncytium formation inhibition assay was performed as described with modifications (navarro et al., ) . in brief, monolayers of vero cells grown in -well plates were infected with hsv- or hsv- at an moi of pfu per cell. after h of adsorption at °c, the virus inoculum was removed and cells were washed with pbs twice then incubated in dmem containing % fbs in the presence of antibodies, m f ( μg/ml) and c ( μg/ml), mouse igg ( μg/ml), or medium alone as a control. after h p.i., cells were fixed and then stained with hematoxylin-eosin (he) staining. the inhibition of syncytium formation was examined by light microscopy. to quantify the inhibition of syntytium formation, the numbers of nuclei in syncytia were counted in randomly chosen area, and the percentage inhibition was determined relative to no antibody treatment group. only syncytia containing or more nuclei were analyzed. three independent experiments were calculated. mouse intravaginal hsv- challenge was established as previously described (cena-diez et al., ) . six-weeks-old female balb/c mice were rendered susceptible to infection by subcutaneous injection with . mg of medoxyprogesterone acetate (sigma) days prior to challenge (parr et al., ) . mice were randomly assigned to five groups of or each. mice were anesthetized with pentobarbital sodium and intravaginally challenged with × pfu/ μl of hsv- . the infected mice were passively immunized by intravenous injection of , , and mg/kg of m f at , , and h after intravaginal hsv- challenge. pbs group was set as control. mice in each group were examined daily for symptoms of genital disease, body weight over days, as well as mortality for days. disease score was graded from to : no apparent infection scored ; genital erythema scored ; moderate genital infection scored ; purulent genital ulceration, hair loss and generally poor condition scored ; severe ulceration of genital and surrounding tissue, and hind limb paralysis scored . mice that developing % loss in body weight, debilitating symptoms of disease or paralysis were sacrificed and considered to have died the following day in all survival analyses. surviving mice were sacrificed at the indicated times after infection. data are presented as means ± standard error of the mean (sem). sequence alignment was performed with dnaman v . statistical analysis was performed using graphpad prism . significant differences were analyzed by log-rank (mantel-cox) test and gehan-breslow-wilcoxon test. p-values were calculated, and statistical significance is reported as highly significant using *(p < . ), ** (p < . ), or *** (p < . ). (original magnification, × ) (c) and (d) virus neutralization assay. hsv- or hsv- ( pfu/well) was incubated with indicated serially diluted antibodies m f, c or preimmune serum. at h p.i., cells were stained and the number of plaques counted. the percentage inhibition was determined relative to the negative control. representative data (mean ± sem) from three independent experiments are shown. to develop functional monoclonal antibody against hsv- gd, we immunized balb/c mice with purified extracellular domain of gd protein. after the primary screening of hybridomas by elisa, western blot analysis and immunofluorescence assay (ifa), a total of strains of positive hybridoma cells were obtained (table s ). epitope mapping of these mabs was determined using the expressed truncated gd proteins, including gd - t, gd - t, gd - t, gd - t, gd - t, gd - t and gd - t, with an overlap of - amino acids between each pair of adjacent fragments (fig. a) , by western blot analysis (data not shown). among these mabs, m f and another mabs recognized truncated gd - t, which is located in the c-terminus (residues - ) of the gd ectodomain (fig. b) . in contrast, c (fig. b ) and the remaining mabs recognized both nterminal fragments, gd - t and gd - t, suggesting that they recognized the overlap region (residues - ) of the two fragments. therefore, two kinds of mab, as represented by m f and c , recognizing different epitopes of hsv- gd, were chosen for further characterization. to characterize the specific recognition ability of m f and c , we first tested their ability to recognize denatured and native forms of hsv- gd and hsv- gd by western blot assay and ifa. western blot analysis revealed that m f reacted with the expected gd band in the sample of both hsv- and hsv- infected vero cells, while c only recognized gd in the hsv- infected vero cells sample, but not the hsv- infected sample ( fig. a) . the ifa results demonstrated that m f detected native forms of gd in the cells infected with either hsv- or hsv- , while c only detected native gd in the cells infected with hsv- (fig. b) . these results suggested that m f could recognize the denatured and native forms of gd of both hsv- and hsv- , while c could only recognize the denatured and native forms of gd of hsv- . to further confirm whether m f and c could neutralize hsv infection, virus neutralization assays were performed. either hsv- or hsv- ( pfu/well) were pre-incubated with serially diluted mabs, m f and c , before infection into vero monolayer cells. pre-immune mouse serum was used as negative control. neutralization assay demonstrated that m f neutralized both hsv- and hsv- in a dosedependent manner, with about ∼ % hsv- infection and about ∼ % hsv- infection being inhibited at an antibody concentration of μg/ml. in contrast, c demonstrated no neutralizing activity of hsv- and low neutralizing activity of hsv- infection groups (only about % virus infection being inhibited at a concentration of μg/ ml) (fig. c and d) . these results suggested that m f but not c , is a neutralizing mab against hsv- and hsv- infection. as shown above, m f had a high level of neutralization activity against both hsv- and hsv- , and m f recognized a linear epitope, gd - t, which is located in the c-terminus of the gd ectodomain. to identity accurately the epitope recognized by m f, we constructed a series of further truncations of the c-terminal region (residues to ) of hsv- gd protein (fig. a) . by western blot analysis, gd - t but not gd - t that could be recognized by m f, indicating that p is the last c-terminal amino acid critical for m f binding (fig. b) . similarly, p is the first n-terminal amino acid critical for m f binding. on this basis, pnwhip was considered to be the precise epitope for m f binding. sequence alignment showed that these residues are highly conserved among hsv- and hsv- (fig. c) . these results explain why m f recognized the gd protein of both hsv- and hsv- . direct transmission between adjacent cells (cell-to-cell spread) is an efficient route for hsv to bypass the host immune system. not all neutralizing mabs can prevent cell-to-cell spread of herpesviruses (co et al., ; hooks et al., ) . therefore, blocking of cell-to-cell spread is considered to be an important aspect in evaluating the in vivo protective efficiency of neutralizing antibodies. we herein analyzed the ability of m f to inhibit cell-to-cell spread of hsv- and hsv- in vitro. confluent bhk cell monolayers were initially incubated with either hsv- or hsv- at an moi of . pfu per cell. to prevent viral spread through viral particles moving across the cell culture supernatant (cellfree spread), bhk cell monolayers were treated with an excess of m f or c , and treatment with mouse igg or culture medium alone was performed as controls. after h p.i., the transmission of virus was detected by immunostaining with mouse polyclonal antibody against gd. as shown in fig. a , m f completely inhibited cell-to-cell spread of both hsv- and hsv- , as evidenced by observing the limited fluorescence caused by virus infection. in contrast, neither c nor mouse igg could sufficiently inhibit transmission of hsv- or hsv- , and extensive virus spread on bhk cell monolayer was observed by immunostaining. when the infected cells were quantified, the results clearly showed that m f is able to inhibit cell-to-cell transmission in hsv-infected cells (fig. b) . to further characterize the mode of action of m f, we analyzed whether this mab could inhibit receptor binding. we compared the neutralization efficacy of m f when the antibody was added before (pre-attachment) or after (post-attachment) hsv virions interacted with vero cells. c was chosen as a negative control. by pre-or postattachment assays, when m f was added before virus-host interaction (pre-attachment), about % hsv- infection was inhibited at a concentration of μg/ml (fig. a) , about % hsv- infection was inhibited at a concentration of μg/ml when m f was added after hsv- virions interacted with vero cells (post-attachment) (fig. b) . almost equal neutralization efficacy of m f was observed, irrespective fig. . m f blocks cell-to-cell spread of hsv- and hsv- . (a) monolayers of bhk cells were infected hsv- or hsv- at an moi of . pfu per cell. cell monolayers were treated with an excess of antibodies, m f and c ( μg/ml each). mouse igg ( μg/ml) or culture medium alone was performed as controls. at h p.i., cells were fixed and observed by fluorescence microscopy. bar represents μm. the experiments were repeated at least twice. (b) the number of infected cell with gfp fluorescence per total cells was calculated in randomly chosen areas. the percentage infection was determined relative to control. the experiment was repeated twice. * p < . , ** p < . , *** p < . . of whether the antibody was added before or after hsv- virions interacted with vero cells. in contrast, the negative control, c , had no neutralizing ability either before or after virions were incubated with cells. the hsv- infection group showed a similar result. about % hsv- infection was inhibited in the pre-attachment treatment (fig. c ) and about % hsv- infection was inhibited in the postattachment treatment with an antibody concentration of μg/ml of m f (fig. d ). in contrast, c had low ( % hsv- infection being inhibited) or no neutralizing ability in the pre-attachment or post-attachment treatments, respectively ( fig. c and d) . the existing data indicated that m f does not interfere with the virus binding process, m f therefore neutralizes hsv entry at the post-binding stage. after binding of gd to a cell surface receptor, it undergoes a conformational change, which is also a key event for triggering later steps leading to fusion. since m f might neutralize hsv entry at the post-binding stage, we asked whether m f could inhibit the virus-induced membrane fusion process. monolayers of vero cells were infected with hsv- or hsv- at an moi of pfu per cell. after h of adsorption at °c, the virus inoculum was removed and cells were washed twice with pbs, the cells were then incubated with an excess of m f, c , mouse igg, or medium alone as a control. after h, syncytium formation was observed by staining with he staining. as shown in fig. a , in both hsv- and hsv- infection groups, obvious syncytia with multinuclear cells were observed when infected cells were treated with preimmune serum, mouse igg or c . however, when cells were incubated with m f, syncytium formation was almost completely inhibited. the syncytium formation inhibition of vero cells with different treatment was quantified in fig. b . this result suggested that m f indeed inhibited the hsv-induced membrane fusion. to investigate the protective efficacy of m f in vivo, we exploited a fig. . m f neutralizes hsv- and hsv- at the post-binding stage. comparison of neutralization efficacy of m f when serial dilutions of antibodies were added before (a and c, preattachment) or after (b and d, post-attachment) hsv virions interacted with vero cells. c was used as a control. neutralization efficacies were determined after h as described above for the standard neutralization assay. the percent inhibition was determined relative to the negative control. representative data (mean ± sem) from at least independent experiments performed are shown. r. du et al. antiviral research ( ) - lethal female balb/c mice model for hsv infection. to assess the therapeutic efficiency of m f, mice were randomly assigned to five groups of or each. in pbs-treated group, intravaginal infection of hsv- ( × pfu/ μl) of six-weeks-old female balb/c mice resulted in rapid progressive disease (fig. a ) and steady decrease of body weight from day post infection (fig. b) , with a median survival time of days (fig. c) . the experimental groups of balb/c mice were treated intravenously with , , , or mg/kg of antibody at , and h after intravaginal hsv- challenge. as shown in fig. , mice receiving mg/kg of m f were slightly protected against lethal infection by hsv- , the body weight of this group decreased as fast as the pbs-treated group, with a median survival time of days longer than those of control mice treated with pbs. however, mice treated with mg/kg or higher concentrations of m f were completely protected from lethal hsv- infection (fig. c) . no symptom of hsv- infection (fig. a ) or loss of body weight was observed among these groups (fig. b) . glycoprotein d (gd) is the most abundant glycoprotein on the virion and the major stimulus for virus-neutralizing antibodies of hsv. for both vaccine design and novel therapeutic strategies, it is important to study epitopes on gd that stimulate virus-neutralizing antibodies. according to the properties of a panel of mabs to gd and gd mutants, which were used to define the relationship between gd structure and function, anti-gd mabs have been previously arranged into groups that recognize distinct discontinuous native and continuous denatured epitopes. most mabs that recognize discontinuous native epitopes have virus-neutralizing activity. for example, mc (which recognizes residues and ), dl (which recognizes residues and ), mc (which recognizes residues - ) and mc (which recognizes residue ) have been reported previously to be neutralizing mabs that recognize discontinuous conformational epitopes. lazear et al., ; muggeridge et al., ; nicola et al., fig. . m f inhibits syncytium formation of hsv- and hsv- . (a) vero cells were infected with hsv- or hsv- at an moi of pfu per cell and then incubated in dmem containing % fbs in the presence of antibodies, m f ( μg/ml) and c ( μg/ml). mouse igg ( μg/ml), or medium alone were used as control. after h, cells were fixed and then stained with he staining. the inhibition of syncytium formation was observed by light microscopy. bar represents μm. the experiments were repeated at least twice. (b) syncytium formation of vero cells with different treatment as shown in panel a was quantified by counting the number of nuclei in syncytia of randomly chosen area. the percentage inhibition was determined relative to no antibody treatment group. only syncytia containing or more nuclei were analyzed. data were representative of three independent experiments. * p < . , ** p < . , *** p < . . ; whitbeck et al., ) . there are also a number of reported mabs that recognize continuous denatured epitopes of hsv gd. among these mabs, most have no or low virus-neutralizing ability, except for the mab d (which recognizes residues to ), which can neutralize viral infectivity by blocking gd-receptor interaction (cairns et al., ) . in this study, we found a novel mab, m f, to be another virusneutralizing mab whose epitope is located within a continuous antigenic determinant. epitope mapping assays indicated that m f recognized conserved residues to which located in the c-terminal pro-fusion domain (residues - ) of gd ( figs. and ) . sequences alignment analysis showed that this epitope is highly conserved among a broad range of type- and type- hsv strains (data not shown). this may also explain why the recognition specificity of m f was type-common. it is likely that m f can be efficacious among divergent isolates yet the breadth of m f neutralizing activity against a panel of hsv- and - isolates in vitro need to be performed. in addition, the resistance evaluation of m f and alanine scanning of - should also be carried out in the future. the recognition specificity of m f was type-common, and m f had a high level of hsv virion neutralizing activity (fig. ) . the epitopes of three reported type-common mabs, bd (which recognizes residues to ), dl (which recognizes residues to ), and bd (which recognizes residues to ), were continuous and also located in the c-terminal pro-fusion domain of gd. however, they had no neutralization activity against both hsv- strain kos and hsv- strain (isola et al., ; lazear et al., ) . previously, hooks and his colleagues found hsv- , human and murine cmv and vaccinia could spread in the presence of neutralizing antibodies generated in rabbits (hooks et al., ) . co et al. humanized two murine monoclonal antibodies against hsv gb and gd, which had been shown to neutralizing hsv- in vitro, however, neither humanized and murine anti-gd mab was able to protect against viral spread. while humanized and murine anti-gb mab protected cells from viral spread (co et al., ) . our research showed that m f is able to inhibit cell-to-cell spread of hsv- and hsv- in vitro (fig. ) , which is an efficient way for hsv to bypass the host immune system through the direct transmission adjacent cells. to our knowledge, m f is the first epitope-mapped neutralizing mab against hsv gd reported to be capable of inhibiting cell-to-cell spread of hsv- and hsv- . antibodies against entry glycoproteins have the ability to neutralize virions via interfering with receptor binding or subsequent fusion events. for example, antibodies targeting both stalk (s ) and globular (s ) subunits of the spike protein of severe acute respiratory syndrome (sars) coronavirus were able to abolish the binding between s protein and its cellular receptors. mabs neutralize hiv by inhibiting the binding of gp to the cd receptor (wibmer et al., ; zeng et al., ) . the above-mentioned neutralizing mabs mc and dl neutralize virus by blocking binding of gd to both receptors, hvem and nectin- . in addition, there are many reports of mabs blocking virus entry at the post-receptor-binding step. for instance, neutralizing mabs specific for the c-terminal domain of the murine leukemia virus (mulv) surface (su) envelope protein subunit interfere with a post-attachment event necessary for membrane fusion (burkhart et al., ) . mab d against the f subunit of the helicoverpa armigera nucleopolyhedrovirus f protein neutralizes virus entry by inhibiting membrane fusion (zou et al., ) . our data from pre-attachment and post-attachment neutralization assays indicated that m f does not interfere with the virus binding process; instead, it might neutralize hsv entry at the post-binding stage (fig. ) . by observing hsv-induced membrane fusion following treatment with m f, we determined that m f could almost completely inhibit syncytium formation (fig. ) , which further confirms that m f could inhibit the membrane fusion event rather than the virus binding process. consistent with current findings, earlier studies (cocchi et al., ; fusco et al., ) established that the profusion domain (residues - ) is required for viral infectivity and fusion but not for receptor binding and the substitutions of some pro residues in pfd (amino acids , , , and ) affected steps in entry and fusion. however, the detailed mechanism of how m f inhibits the hsv fusion process needs further study. based on the high level of hsv virion neutralizing activity, we also investigated whether application of m f could confer protection in vivo by exploiting a lethal hsv- intravaginally infected female balb/c mouse model to evaluate the potency of m f as therapeutic alternative to counteract hsv infection. our data demonstrated that mab m f conferred protection in a dose-dependent manner and that mg/kg or more of m f provided full protection (fig. ) . consistently, intracutaneously injection human recombinant neutralizing mab hsv specific for gd (type-common and its epitope located in residues to ) can protect mice effectively when administered systemically zeitlin et al., ) . passive immunization of immunocompetent animals with gd specific mab hd (a type-common fig. . m f protects mice from lethal hsv- infection. (a) symptoms of genital disease in each group was scored daily as described in the text for days after intravaginally hsv- challenge. data was shown as the mean values of mice in each group. (b) mice were weighed daily and body weight are expressed as a percentage of the value prior to treatment. data represent mean ± standard error. (n = animals per group for pbs; n = for all other groups) (c) survival of mice in each group monitored for indicated days. (n = animals per group for pbs; n = for all other groups). data were analyzed by log-rank (mantel-cox) test and gehan-breslow-wilcoxon test. * p < . , *** p < . . neutralizing mab, and its discontinuous epitope located in residues to ) postexposure at appropriate times demonstrated protection against hsv- strains (htz, mp, and a mutant strain mp) and hsv- strain g -induced neurological disease (dix et al., ; pereira et al., ) . furthermore, intravaginally administered murine anti-gb mab post hsv- infection conferred full protection in an immunodeficient non-obese diabetic (nod)/severe combined immunodeficiency (scid) mouse model (krawczyk et al., ) . these results encourage further potency of neutralizing mabs for clinical therapy against severe hsv diseases. in conclusion, our results have demonstrated for the first time that mab m f targeting a new continuous epitope (residues to ) within the pro-fusion domain has a high level of virus-neutralizing activity. these finding will enrich 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papillomavirus cofactor in the etiology of invasive cervical cancer update on herpes virus infections of the nervous system the major neutralizing antigenic site on herpes simplex virus glycoprotein d overlaps a receptor-binding domain hiv broadly neutralizing antibody targets topically applied human recombinant monoclonal igg antibody and its fab and f(ab') fragments protect mice from vaginal transmission of hsv- quantitative comparison of the efficiency of antibodies against s and s subunit of sars coronavirus spike protein in virus neutralization and blocking of receptor binding: implications for the functional roles of s subunit a novel multiplex real-time pcr assay for the detection and quantification of hpv / and hsv / in cervical cancer screening characterization of two monoclonal antibodies, f and d , against the major envelope fusion protein of helicoverpa armigera nucleopolyhedrovirus we thank members of our laboratory for discussions and the core facility and supplementary data related to this article can be found at http://dx. doi.org/ . /j.antiviral. . . . key: cord- -qos vu r authors: dejnirattisai, wanwisa; webb, andrew i.; chan, vera; jumnainsong, amonrat; davidson, andrew; mongkolsapaya, juthathip; screaton, gavin title: lectin switching during dengue virus infection date: - - journal: j infect dis doi: . /infdis/jir sha: doc_id: cord_uid: qos vu r dengue virus receptors are relatively poorly characterized, but there has been recent interest in c-type lectin molecules, dendritic cell–specific intercellular adhesion molecule (icam- )–grabbing nonintegrin (dc-sign) and its close homologue liver/lymph node–specific icam- –grabbing integrin (l-sign), which can both bind dengue and promote infection. in this report we have studied the interaction of dengue viruses produced in insect cells, tumor cell lines, and primary human dendritic cells (dcs) with dc-sign and l-sign. virus produced in primary dcs is unable to interact with dc-sign but remains infectious for l-sign–expressing cells. skin-resident dcs may thus be a site of initial infection by insect-produced virus, but dcs will likely not participate in large-scale virus replication during dengue infection. these results reveal that differential glycosylation of dengue virus envelope protein is highly dependent on cell state and suggest that studies of virus tropism using virus prepared in insect cells or tumor cell lines should be interpreted with caution. the global prevalence of the dengue virus (denv) has grown dramatically in recent decades, and it is now endemic in . countries, with some . billion people at risk of infection. dengue is an arthropod-borne flavivirus that can be subdivided into the major serotypes (den- -den- ). most dengue infections either are asymptomatic or lead to a self-limiting febrile illness, dengue fever. in some cases the illness is more severe, leading to dengue hemorrhagic fever (dhf) with severe plasma leakage and bleeding that can be life threatening. dhf is more common in individuals undergoing secondary heterologous dengue infection than those suffering primary infections. there has been considerable work to understand the mechanism of severe disease, but the increased frequency during secondary infection and the occurrence of severe symptoms at a time when virus loads are falling sharply imply that it likely results from immunopathology driven by the acquired immune responses, rather than from direct viral cytopathology [ ] [ ] [ ] [ ] . levels of a number of cytokines, such as interferon c and tumor necrosis factor a [ , ] , have been shown to correlate with disease severity, and a storm of inflammatory cytokine secretion has been proposed to lead to the vascular leak characteristic of dhf. recent reports have identified monocytes as a major cell target of viral replication, and heparin sulfates, dc-sign, mannose receptor, and other glycoproteins have been proposed as cellular receptors for denv [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . dc-sign polymorphism has been shown to be associated with the disease severity [ ] . clec a has also recently been described as a proinflammatory receptor for denv that contributes to lethal disease in a mouse model [ ] . as dengue virus circulates between hosts, humans and insects, it has to be adapted to replicate and infect both species. the majority of studies of dengue infection and tropism use viruses produced in insect cell lines such as c / or mammalian tumor cell lines such as vero cells. infection of humans occurs in a stepwise fashion: initial infection of cells with insect virus, followed by sequential infection of cells by mammalian-produced virus. we were interested to determine whether there were any differences in the tropism of viruses produced in primary nontransformed human cells. the dengue- strain, , was grown in c / cells, vero cells, and monocyte-derived dendritic cells (dcs). cell-free supernatants were used either neat or after concentration by ultracentrifugation at , rpm for h at °c, and the virus pellet was resuspended in . % fetal bovine serum (fbs)/ leibovitz l- . to concentrate large volumes of low-titer denv supernatant, denv were precipitated with % polyethylene glycol (sigma) before ultracentrifugation. u were maintained in % fbs/roswell park memorial institute medium (rpmi). nih/ t , and dc-sign and l-sign nih/ t cell lines were obtained through the aids research and reference reagent program (division of aids, national institute of allergy and infectious diseases, from drs thomas d. martin and vineet n. kewal ramani) and maintained in % fbs/ dulbecco's modified eagle's medium. all media were supplemented with mmol/l l-glutamine, u/ml penicillin, and u/ml streptomycin. the viral titers were determined by a focus-forming assay. briefly, virus was serially diluted and incubated with vero cells for hours at °c. the monolayers were then overlaid with . % carboxymethylcellulose and incubated at °c for days. virus foci were stained with anti-e antibody followed by peroxidase-conjugated anti-mouse immunoglobulin (ig) and visualized by the addition of , #-diaminobenzidine tetrahydrochloride substrate. dcs were cultured as described elsewhere [ ] . human cd monocytes were cultured in rpmi with ng/ml rhugm-csf (first link) and ng/ml rhuil- (ebioscience). the appropriate phenotype of immature dc (ie, lacking cd but expressing major histocompatibility complex class i and class ii and dc-sign) was confirmed. cells were infected with denv at a multiplicity of infection (moi) of for h. cells were washed twice in facs wash (fw; phosphate-buffered saline [pbs] containing . % bovine serum albumin, % fbs, % human serum, . % nan ). cells were fixed with % paraformaldehyde/pbs for minutes and permeabilized with . % saponin in fw for minutes. cells were then incubated with anti-ns mab or with anti-e mab followed by anti-mouse igg/pe (dakocytomation) in . % saponin/fw. cells were resuspended in fw and analyzed by facscan (becton dickinson). data were analyzed by using flowjo software (tree star). t and t were incubated with u/ml heparin for minutes at room temperature before being infected with denv. cells were incubated with denv produced from different cell types at equal amounts of e protein (measured by enzymelinked immunosorbent assay) for h at room temperature. after washing with ice-cold fw, cells were fixed with % paraformaldehyde/pbs, and surface-bound virus was detected with an anti-e mab and anti-mouse igg/pe followed by facs analysis. the pcdna -ll-sign plasmids expressing (n ) and (n ) tandem neck region repeats or a mixture of n and n plasmids (ratio : ) were transfected into t cells using the fugene (roche). l-sign expression was verified with l-sign-specific antibody (clone , r&d systems). denv supernatant was precleared with protein a-agarose for one h at °c. bead-free supernatant was incubated with lg of g at °c for h followed by protein a-agarose for h. the beads were washed with . % tween/pbs times and eluted with nonreducing loading buffer. the sample was run on nonreducing % sodium dodecyl sulfate (sds) polyacryramide gels and electroblotted onto nitrocellulose membrane (amersham). glycan types on denv proteins were determined using the dig glycan differentiation kit (roche). endoglycosidase h (endo h) or n-glycosidase f (pngase f; new england biolabs) was performed as described elsewhere [ ] . digested proteins were separated by % sds polyacryramide gels and analyzed by western blot. e protein was detected by anti-e mab followed by peroxidase-conjugated anti-mouse igg ab. following the bite from an infected mosquito, the host first encounters virus produced in the mosquito, and following this initial inoculation subsequent rounds of infection are driven by virus produced by host cells. to study these distinct stages of pathogenesis, we compared viruses from c / (insect cells) and virus produced from primary human monocyte-derived dendritic cells. viral supernatants were titered using a focus-forming assay on vero cells, and equal amounts of titered virus were used to infect vero, t, or dcs at an moi of . the percentage of infected cells was monitored by cytofluorometry staining of the intracellular nonstructural dengue antigen ns , which is produced only following productive infection. insect-derived virus was equally competent at infecting the cell types with high efficiency ( figure a-c) . surprisingly, dc-produced virus was not able to reinfect dcs but was nevertheless fully competent at infecting t and vero cells. the lack of infectivity of dc-produced virus on dcs was also shown using a different mab, g , which reacts with an epitope on dengue envelope protein ( figure c , lower panel). a time course of infection was performed where dcs or vero cells were infected with virus produced in c / , vero, or dcs, and infection was monitored by facs or using a focus-forming assay to measure infectious virus harvested from the supernatants of infected cells ( figure d -e). at , , and h the dc-produced virus showed a much reduced ability to infect dcs when compared with virus produced in c / or vero cells. the experiments we have described above used the dengue serotype strain . to see whether these results could be generalized, we checked infection of both dcs and vero cells with dengue serotype strain new guinea c, serotype strain hawaii, serotype strain h , and serotype strain h . infection efficiency was measured by facs at and hours following infection with virus produced in either c / cells or dcs (figure a-b) . in all cases dc-produced virus was much less infectious for dcs than insect-produced virus, whereas infection of vero cells was roughly equal. it is known that mature dcs are relatively resistant to dengue infection, so to rule out the possibility of any dc-produced cofactors that might induce maturation or any other form of resistance to infection we performed a mixing experiment whereby dc-produced and c / -produced viruses were used to infect dcs. in these experiments using mixed viruses the dcs were again susceptible to infection, presumably by the c / produced virus fraction ( figure c ). the lack of reinfection of dcs by dc-produced virus may reflect a difference in the surface binding interaction of dc-versus insect-produced viruses to dcs. to test this vero cells and dcs were incubated with denv produced from the different sources, and binding to the cells was then assessed by staining with the anti-e mab g . virus produced in c / cells could bind to dcs, while binding of dc virus back onto dcs was almost completely absent ( figure d ). conversely, both dc-and insect-produced viruses were able to bind to vero cells. to formally prove that the loss of infection of dcs was a result of the loss of affinity of dc-produced virus for dc-sign, we went on to test infection on t cells expressing dc-sign and included in these assays the related c-type lectin l-sign ( figure a ), which has also been reported to be a receptor for dengue virus. insect-derived virus could efficiently infect both dc-sign-and l-sign-expressing cells when compared with control t cells ( figure b ). similar to the lack of observable infection of dcs, the dc-derived virus showed a much lower level of infection on dc-sign-expressing t cells, with , % infection. surprisingly, however, this virus progeny was still able to infect cells expressing l-sign, with up to % infection observed. previous reports with dengue virus have suggested that the virus shows equal tropism for both dc-sign and l-sign. these reports were from virus produced in tumor cell lines and not from primary cells. we tested the infectivity of virus produced in vero cells, and in agreement with these previous reports dengue produced in this tumor cell line was able to efficiently infect t cells via either dc-sign or l-sign ( figure c ). finally, we tested the binding of insect-and dc-produced virus to the t transfectants ( figure d ). in agreement with the dc binding experiments dc-produced virus was unable to bind to dc-sign-expressing dcs, whereas both insect-and dc-derived viruses could bind to l-signexpressing cells. to gain insight into the glycosylation profiles of denv produced in c / , vero, and dc, purified virus was tested against a panel of lectins with differing carbohydrate-binding specificities by western blot ( figure a ). among the plant lectins tested, gna is the only one that selectively interacts with high-and pauci-mannose-type n-glycans. sna and maa bind to sialic acid terminally linked to galactose by a , linkage and a , linkage, respectively, which may occur on complex-type n-glycans. dsa recognizes repeating n-acetyllactosamine (galactosyl b , n-acetylglucosamine) sequences; these may also occur on complex-type n-glycans. pna, unlike dsa, binds preferentially to b , -linked terminal galactose, such as galactosyl b , n-acetylgalactosamine, a sequence commonly occurring on o-glycosylated proteins and gangliosides. virus produced in c / cells bound exclusively to gna, implying that it contained predominantly high-or pauci-mannose n-glycans consistent with the glycosylation patterns seen in insect cells. vero-produced virus was bound by of the lectins tested-gna, sna, and dsa-suggesting that the virus contained a variety of high-mannose and complex-or hybrid-type n-glycans with or without a , -linked sialic acids, possibly on polynacetyllactosamine outer chains. the dc-produced virus was bound only by sna and dsa, indicating that there was a lack of high-mannose or hybrid n-glycans and that only complex-type n-glycans were present. all of the lectin binding signals occurred at the same position on the gels as e protein in the region of kda; we did not see any signal at kda where prm would be expected to migrate. however, this could be due to a low level of prm on these viruses. finally, we assessed the n-linked glycan on the envelope protein by digestion with either endo h, which will remove high-mannose simple glycans containing or more terminal mannose residues, or pngase f, which removes all simple or complex n-linked glycan ( figure b ). dc-produced virus showed a mobility shift of around or kda corresponding to the addition of or complex carbohydrates at positions and . this was completely resistant to digestion with endo h, indicating the presence of complex low-mannose carbohydrate. for both insect-and vero-produced virus, envelope protein migrating at or kda higher than the pngase f-digested material indicated, as with dc-produced virus, that envelope had either or added sugars. following endo h digestion a complex pattern of bands was revealed, indicating that some of the added sugars were endo h sensitive and therefore contained . terminal mannose residues, whereas others were endo h resistant and contained more heavily processed structures, which is in agreement with a recent report [ ] . we conclude from these experiments that in c / , vero cells, and dcs both n-linked glycosylation sites can be used but that a single site is used in % of cases. envelope from dc-produced virus contains complex, highly processed endo h-resistant carbohydrate. however, in both c / and vero, a proportion of the sugar is high mannose and therefore will allow interaction with dc-sign, which is consistent with the results from lectin blotting shown above. l-sign contains a tandem repeat in its neck region, nterminal to the carbohydrate recognition domain, which can be of variable length between and repeats. l-sign exists as tetramers, and previous studies have suggested that heterogeneity in the tandem repeat region of the l-sign neck region may contribute to severe acute respiratory syndrome (sars) susceptibility by altering the viral env-receptor affinity [ ] . to determine whether heterogeneous l-sign neck lengths affect denv infection levels, we examined the effects of a single length repeat and compared it with cells expressing different-length l-sign alleles simultaneously. we examined this by transient transfection, and t cells were used for these assays as they can be transiently transfected to a high level. t were transfected with l-sign containing or repeats singly or together in equal amounts. l-sign expression was confirmed by surface staining and flow cytometry. equal expression of alternate-length l-sign constructs was also confirmed by western blotting. transfectants were then infected with dengue virus, and infection was monitored by intracellular staining together with an antibody specific to l-sign to reveal transfected cells. all transfectants were equally infected with no reduction in cells expressing both l-sign alleles, suggesting that heterotetrameric l-sign was as effective as homotetrameric l-sign at promoting infection ( figure ). finally, although dc-produced virus cannot reinfect dcs efficiently, we were interested to determine whether dc-produced virus was still able to infect cells by antibody-dependent enhancement (ade), which would allow it to replicate by infection of fc receptor-expressing cells. c / -and dc-derived viruses were incubated with increasing levels of pooled convalescent dengue immune serum and subsequently used to infect u , a monocyte cell line that expresses the fc receptor and which shows relatively low infectivity without the presence of enhancing antibodies. viruses produced in both dcs and insect cells were susceptible to enhancement, over the same range of antibody concentrations, showing that dc-produced virus could exploit ade to replicate in individuals undergoing a secondary dengue infection ( figure a ). in a final series of experiments we investigated whether dc-produced virus could be induced to infect primary human dcs by ade ( figure b ). dc-produced virus could be enhanced to infection by . -fold, whereas the already high level of infection of dcs by insectproduced virus was not enhanced further by dengue serum. during natural dengue infection, mosquito saliva containing denv particles is injected into the skin during a blood meal. skin-resident immature dcs have been proposed to be the primary site of infection for insect-derived denv [ ] . the primary receptor on dcs for dengue virus is believed to be dc-sign. dc-sign binds n-linked high-mannose oligosaccharides, including glycans with terminal fucose residues that include the blood group lewis x and lewis a eptitopes [ ] . l-sign, like dc-sign, binds to intercellular adhesion molecule (icam- ) and is thought to establish cellular interactions with icam- -expressing t cells. l-sign is able to capture a variety of viruses ( [ ] [ ] [ ] [ ] [ ] . dc-sign and l-sign preferentially bind pyranose sugars, in particular mannose. a study comparing l-sign-and dc-sign-specific ligands using glycan arrays revealed a restricted repertoire of glycan ligands for l-sign, namely, the high-mannose-type n-glycans. in contrast, dc-sign bound to fucosylated glycans in addition to highmannose-type n-glycans [ ] . the dengue e protein has potential n-linked glycosylation sites at positions and . cryo-electron microscopy reconstructions of dengue virus complexed to soluble dc-sign show interaction with the glycan at position [ ] . there have been several reports on the n-linked glycosylation of dengue envelope. some reports suggest the use of both sites, whereas others suggest that only one is used [ , [ ] [ ] [ ] . we show that in c / , vero cells, and dcs either one or both sites are used for the dengue serotype . although both glycosylation sites can play important roles in infectivity and viral replication, functional studies have confirmed the importance of asn- for infection of dc-sign-expressing cells [ ] . wnv e protein contains a single n-linked glycosylation site at position that is absent from some virus strains [ ] . both dengue and wnv contain a single n-linked site in prm, and although prm is cleaved by furin during viral maturation, a substantial fraction of uncleaved prm is found on some dengue viruses, particularly that produced in insect cells, and such partially cleaved viruses can still be infectious. for wnv the n-linked site on prm can also mediate interaction with l-sign, and in common with glycan at position this also showed differential specificity for l-sign when expressed in mammalian cell lines, perhaps mediated by the presence of n-acetylglucosamine-terminated structures [ ] . the difference in the binding specificities of dengue viruses produced in vero versus primary dendritic cells was somewhat surprising and likely related to the expression of high-mannose moieties in vero. a number of tumor cell lines express high-mannose sugars, and we have previously described the generation of a monoclonal antibody that recognizes a variety of tumor cell types and which binds to high-mannose moieties [ ] . the differential affinity of dc-sign for ligands expressed by tumor cell lines versus primary cells has been observed before and led some to speculate that dc-sign may participate in tumor surveillance [ ] . there appears to be a further added level of complexity as the glycosylation profiles may vary in a given cell line depending on the exact position of the n-linked site within the polypeptide chain [ ] . in humans, l-sign expression is restricted to endothelial cells beneath the subcapsular sinus in lymph nodes [ ] , sinusoidal endothelial cells in the liver [ , ] , alveolar and endothelial cells in the lung [ ] , and capillaries in the villous lamina propria of terminal ileum and peyer patches [ ] . dengue antigens have been demonstrated in the sinusoidal tissue of the liver and vascular endothelium of the lung and spleen and may provide an explanation for the unique pathology observed in these organs [ , ] . the accumulation of antigen in l-signexpressing tissue may also result in an increase in localized transinfection in a similar way that l-sign is thought to be responsible for the capture of hcv from the blood and transmission to hepatocytes or in the case of hiv, to cd t cells [ , ] . we conclude that skin-resident dcs are a likely target for initial infection by dengue virus injected by the infecting mosquito; however, subsequent dissemination of the virus to monocytes and other cell types will no longer use dc-sign as a primary receptor and may rely in part on a shift to l-sign as the primary lectin receptor. the differential glycosylation of the denv e protein during replication in primary mammalian cells suggests that studies of virus tropism using virus prepared in insect cells or tumor cell lines should be interpreted with caution. cross-reacting antibodies enhance dengue virus infection in humans immunodominant t-cell responses to dengue virus ns are associated with dhf immunopathological mechanisms in dengue and dengue hemorrhagic fever original antigenic sin and apoptosis in the pathogenesis of dengue hemorrhagic fever multiplex cytokine profile from dengue patients: mip- beta and ifn-gamma as predictive factors for severity high levels of stnfr p and tnf alpha in dengue-infected patients monocytes, but not t or b cells, are the principal target cells for dengue virus (dv) infection among human peripheral blood mononuclear cells phenotyping of peripheral blood mononuclear cells during acute dengue illness demonstrates infection and increased activation of monocytes in severe cases compared to classic dengue fever dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate dc-sign (cd ) mediates dengue virus infection of human dendritic cells the mannose receptor mediates dengue virus infection of macrophages dengue virus binding to human hepatoma hepg and simian vero cell surfaces differs dengue virus entry into liver (hepg ) cells is independent of hsp and hsp heat shock protein and heat shock protein are components of dengue virus receptor complex in human cells identification of grp (bip) as a liver cell expressed receptor element for dengue virus serotype bacterial lipopolysaccharide inhibits dengue virus infection of primary human monocytes/macrophages by blockade of virus entry via a cd -dependent mechanism dendritic cell-specific intercellular adhesion molecule -grabbing non-integrin (dc-sign)-mediated enhancement of dengue virus infection is independent of dc-sign internalization signals dendritic-cell-specific icam -grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses a variant in the cd promoter is associated with severity of dengue disease clec a is critical for denguevirus-induced lethal disease a complex interplay among virus, dendritic cells, t cells, and cytokines in dengue virus infections histidine in the dengue virus type m protein has an important role in virus assembly n-linked glycans on dengue viruses grown in mammalian and insect cells homozygous l-sign (clec m) plays a protective role in sars coronavirus infection human skin langerhans cells are targets of dengue virus infection the dendritic cell-specific c-type lectin dc-sign is a receptor for schistosoma mansoni egg antigens and recognizes the glycan antigen lewis x a dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin (dc-sign)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes hiv- infection l-sign (cd l) and dc-sign (cd ) mediate transinfection of liver cells by hepatitis c virus the location of asparagine-linked glycans on west nile virions controls their interactions with cd (dendritic cell-specific icam- grabbing nonintegrin) human cytomegalovirus binding to dc-sign is required for dendritic cell infection and target cell trans-infection dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus structural basis for distinct ligandbinding and targeting properties of the receptors dc-sign and dc-signr cryo-em reconstruction of dengue virus in complex with the carbohydrate recognition domain of dc-sign the envelope glycoproteins of dengue and dengue viruses grown in mosquito cells differ in their utilization of potential glycosylation sites both e protein glycans adversely affect dengue virus infectivity but are beneficial for virion release essential role of dengue virus envelope protein n glycosylation at asparagine- during viral propagation envelope protein glycosylation status influences mouse neuroinvasion phenotype of genetic lineage west nile virus strains enhanced immune recognition of cryptic glycan markers in human tumors the b homolog butyrophilin btn a is a novel ligand for dc-sign dynamic populations of dendritic cell-specific icam- grabbing nonintegrin-positive immature dendritic cells and liver/lymph node-specific icam- grabbing nonintegrin-positive endothelial cells in the outer zones of the paracortex of human lymph nodes dc-signr, a dc-sign homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans cd l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus expression of dc-sign by dendritic cells of intestinal and genital mucosae in humans and rhesus macaques lethal antibody enhancement of dengue disease in mice is prevented by fc modification localization of dengue virus in naturally infected human tissues, by immunohistochemistry and in situ hybridization we thank t. feizi and yan liu for advice and critically reading the manuscript. key: cord- -gs celr authors: tan, yee‐joo; lim, seng gee; hong, wanjin title: regulation of cell death during infection by the severe acute respiratory syndrome coronavirus and other coronaviruses date: - - journal: cell microbiol doi: . /j. - . . .x sha: doc_id: cord_uid: gs celr both apoptosis and necrosis have been observed in cells infected by various coronaviruses, suggesting that the regulation of cell death is important for viral replication and/or pathogenesis. expeditious research on the severe acute respiratory syndrome (sars) coronavirus, one of the latest discovered coronaviruses that infect humans, has provided valuable insights into the molecular aspects of cell‐death regulation during infection. apoptosis was observed in vitro, while both apoptosis and necrosis were observed in tissues obtained from sars patients. viral proteins that can regulate apoptosis have been identified, and many of these also have the abilities to interfere with cellular functions. occurrence of cell death in host cells during infection by other coronaviruses, such as the mouse hepatitis virus and transmissible porcine gastroenteritis virus, has also being extensively studied. the diverse cellular responses to infection revealed the complex manner by which coronaviruses affect cellular homeostasis and modulate cell death. as a result of the complex interplay between virus and host, infection of different cell types by the same virus does not necessarily activate the same cell‐death pathway. continuing research will lead to a better understanding of the regulation of cell death during viral infection and the identification of novel antiviral targets. the regulation of cell death during a viral infection is an important determinant in the struggle between virus and host for survival. two forms of cell death have been extensively described, one is necrosis, defined as a passive and non-physiological type of death caused by accidental and acute damage to the cell, and the other is apoptosis, defined as an active and genetically regulated process of cell suicide by which an organism eliminates senescent, abnormal and potentially harmful cells. these two forms of cell death are distinguishable by their morphological and biochemical effects on the cell. however, recent studies have shown that necrosis is also highly regulated and can play essential roles in maintaining homeostasis in healthy cells as well as in the elimination of infectious pathogens (assuncao guimaraes and linden, ; nelson and white, ; festjens et al., ; zong and thompson, ) . a novel coronavirus (termed as severe acute respiratory syndrome coronavirus, sars-cov) was the cause of a viral outbreak which caused profound disturbances worldwide in (fouchier et al., marra et al., ; peiris et al., ; rota et al., ) . coronaviruses are a family of enveloped, single-, positive-stranded rna viruses with very large genomic size of~ kb and have been known to infect many animal species as well as humans (siddell, ) . one of the most common abnormalities in sars-cov-infected patients is lymphopenia (peiris et al., ; chng et al., ; chen et al., ) , which could be caused by the depletion of t lymphocytes by apoptosis. indeed, several laboratories have successfully detected sars-cov in the lymphocytes isolated from infected patients, suggesting that the virus can infect lymphocytes (wang et al., ; gu et al., ) . however, there is still no evidence that infection of lymphocytes is the direct cause for lymphopenia in sars patients. in addition, both apoptosis and necrosis have been observed in various infected tissues obtained during autopsy studies on sars casualties (ding et al., ; lang et al., ; chau et al., ; chong et al., ; wei et al., ) . thus, cell death has been observed during sars-cov infection in vivo. this review summarizes current knowledge on the molecular aspects of cell-death regulation during sars-cov infection and the contributions of viral proteins and viral-host interactions to this process. together with studies on other coronaviruses, these investigations provide important insights into the regulation of apoptosis and necrosis during viral infection and contribute to the development of antiviral therapeutics. the main cause of death among sars casualties was respiratory failure as a result of severe lung injury. histopathological examinations revealed extensive damages to the alveolar and bronchial epithelial cells and macrophages, and these are likely to be caused by multiple factors, including cytopathic effects mediated by replication of the sars-cov and the overproduction of immune mediators (see a recent review by chen and subbarao, ) . besides lymphopenia (as described above), there is currently a lack of information on the role of cell death during the earlier stages of infection, as most of these data were obtained during autopsy studies on fatal cases and would therefore reflect the terminal stages of the disease. extra-pulmonary spreading of the virus has also been reported, and in some of these organs, apoptosis and necrosis have been observed. in one study, extensive apoptosis was observed in the hepatocytes of three sars patients who had liver impairment, suggesting that liver damages in these patients may be mediated by apoptosis (chau et al., ) . apoptosis was also observed in the thyroid glands obtained from five fatal sars cases, suggesting that pathogenesis in the thyroid glands may be related to apoptosis induction (wei et al., ) . in various studies, necrosis was also observed in lymphoid tissues and lymph nodes (ding et al., ; lang et al., ; gu et al., ) . the occurrence of apoptosis during sars-cov infection in vitro (i.e. in cell culture systems) has been reported by several groups (mizutani et al., ; tan et al., ; yan et al., ; ren et al., ; bordi et al., ) . in these studies, the vero cell line (or a subclone of vero known as vero e ), which is a green monkey kidney cell line that supports sars-cov replication and shows extensive cytopathic effects upon infection, was used. the induction of apoptosis was dependent on viral replication and could be inhibited by caspase inhibitors or the overexpression of the pro-survival protein, bcl- (ren et al., ; bordi et al., ) . although necrosis was not observed in sars-cov-infected vero e cells (yan et al., ) , it has been observed in different tissues obtained from sars-cov-infected patients (ding et al., ; lang et al., ; chong et al., ) . it is not clear whether necrosis in these tissues represented secondary necrosis reflecting the degradative changes that apoptotic cells undergo at the later stages of apoptosis, but at least one sars-cov protein [open reading frame (orf) b] has been shown to induce necrosis (khan et al., ) . apoptosis and necrosis were also observed during the infection of vero cells by the infectious bronchitis virus (ibv), an avian coronavirus (liu et al., ) . interestingly, necrosis was also observed during infection and could be a more dominant factor for viral-induced cell death, as neither the death of infected cells nor the productive replication of ibv was severely affected by the inhibition of apoptosis by the general caspase inhibitor, z-vad-fmk. like the orf b protein of sars-cov, the orf b protein of ibv is localized to the nucleus (shen et al., ) , although it has not yet been determined whether the latter can induce apoptosis or necrosis. for another human coronavirus, oc , intracerebral inoculation into mice resulted in acute encephalitis, with neuronal cell death caused by both necrosis and apoptosis (jacomy et al., ) . however, infection of mrc- , diploid human fetal lung cells, seems to induce mainly apoptosis (collins, ) . similarly, infection of monocytes/ macrophages in vitro by e, yet another human coronavirus, caused mainly apoptosis, although a few necrotic cells were also observed (collins, ) . in two independent studies, it was demonstrated that the inhibition of apoptosis, either by caspase inhibitors or by overexpression of the bcl- protein, did not affect sars-cov replication in vero cells (ren et al., ; bordi et al., ) , suggesting that apoptosis does not play a role in facilitating viral release. however, this was only performed in the vero cell line, and it is not known whether the inhibition of apoptosis will affect sars-cov replication in other cell lines or animal models. the regulation of cell death in other cell lines may be dramatically different, as some cell lines supported sars-cov replication but, unlike the vero cell line, displayed minimal cytopathic effects (gillim-ross et al., ; kaye et al., ) . analysis of host gene transcriptions in various cell lines also revealed significant differences in cellular responses to sars-cov infection. for example, tang et al. ( ) reported that the upregulation of pro-apoptotic genes in sars-cov-infected huh cells, while the opposite was observed in sars-cov-infected intestinal cell lines, caco- and cl- (cinatl et al., ) . besides these two studies, the transcriptional profiles of apoptosis-related genes in sars-cov-infected vero e have also been reported (leong et al., ) . interestingly, several pathways that promote apoptosis, as well as those that prevent apoptosis, appeared to be modified during sars-cov infection, suggesting that the cell death may be cell death during infection by coronaviruses regulated differently at different stages of the sars-cov life cycle. similar results were obtained when gene profiling was performed using peripheral blood mononuclear cells (pmbcs) from healthy donors that were inoculated in vitro with sars-cov (ng et al., ) . other studies that used pmbcs isolated from sars patients also revealed changes in the transcription of many genes involved in cell-death regulation (reghunathan et al., ; yu et al., ; shao et al., ) . one interesting gene that was found to be upregulated is lipocalin , which belongs to a class of secreted proteins that are thought to trigger apoptosis in immune cells via an unknown cell receptor (reghunathan et al., ) . the authors speculated that the upregulation of lipocalin is a host response to limit tissue damage and inflammation and the overexpression of lipocalin could lead to lymphopenia in sars patients. the sars-cov genome has the typical organization as other members of the coronaviridae family (marra et al., ; rota et al., ) . the first two-thirds of the sars-cov genome encodes the replicase polyproteins (pp a and pp ab) that are processed to yield non-structural proteins, some of which are responsible for replicating the viral genome and/or generating a nested set of subgenomic mrnas to express all the other orfs in the genome (ziebuhr, ) . the orfs for the main structural proteins, spike (s), envelope (e), membrane (m) and nucleocapsid (n), are encoded in the remaining portion of the genome, and interspaced between these are the orfs for eight putative accessory proteins (i.e. orfs a, b, , a, b, a, b and b). while the sars-cov replicase and structural proteins share some degree of sequence homology with those of other coronaviruses, the accessory proteins do not show significant homology to viral proteins of known coronaviruses (tan et al., ) . the overexpression of some of the sars-cov proteins could induce apoptosis and/or necrosis, and this is summarized in table . while the c-like protease (also known as cl pro or m pro ) is the only replicase gene product that has been shown to induce apoptosis (lin et al., ) , all the four main structural proteins (s, e, n and m) could induce apoptosis (surjit et al., ; chow et al., ; yang et al., ; zhao et al., ) . as shown in table , the experiments were performed in one or two cell lines (table ) , and it has not been demonstrated whether the induction of apoptosis by these structural proteins is cell line-specific. it is difficult to compare the apoptosis-inducing capabilities of the structural proteins, as different laboratories have used different cell lines for their investigations. for example, the apoptosis induction by the e protein was demonstrated in jurkat t cells , while the apoptosis induction by the n protein was demonstrated in cos- cells (surjit table activates the transcription factor nf-kb (lin et al., ) . vero e cell line (chow et al., ) upregulates the expression of cox- (liu et al., a) . jurkat t cell line alters the membrane permeability of mammalian cells (liao et al., ) . forms cation-selective ion channels in planar lipid bilayers (wilson et al., ) . human pulmonary fibroblast (zhao et al., ) not known. nucleocapsid cos- cell line (surjit et al., ) ; human pulmonary fibroblast (zhao et al., ) upregulates the jnk and p mapk pathways (surjit et al., ) . downregulates erk, phospho-akt and bcl- (surjit et al., ) . inhibits the activity of cyclin-cyclin-dependent kinase complex and blocks s phase progression (surjit et al., ) . activates the transcription factors, nf-kb and ap- (he et al., ; liao et al., ) . upregulates the expression of cox- (yan et al. ). orf a vero e cell line (law et al., ) forms ion channel in xenopus oocytes (lu et al., ) . activates the transcription factor nf-kb (kanzawa et al., ) . orf b cos- cell line ; vero e cell line (khan et al., ) induces cell cycle arrest at the g /g phase . localizes to the mitochondria (yuan et al., a) . orf a hela, hepg , a , t, cos- and vero e cell lines ; a and t cell lines (kopecky-bromberg et al., ) inhibits cellular protein synthesis (kopecky-bromberg et al., ) . induces the phosphorylation and activation of p mapk (kopecky-bromberg et al., ) . blocks cell cycle progression at g /g phase via the cyclin d /prb pathway (yuan et al., b) . activates the transcription factor nf-kb (kanzawa et al., ) . nf-kb, nuclear factor kappa b; cox- , cyclooxygenase- ; jnk, c-jun n-terminal kinase; p mapk, p mitogen-activated protein kinase; erk, extracellular-signal-regulated kinase; ap- , activator protein . et al., ) and human pulmonary fibroblast (zhao et al., ) . it is also interesting to note that some of the structural viral proteins (e, n and m) induce apoptosis only in the absence of growth factors. this may imply that the induction of apoptosis by these proteins can only occur after the host cells become stressed at later stages of infection. further studies are required to explore this possibility and define the precise mechanisms for cell-death induction. three of the accessory proteins, orfs a, b and a, have also been shown to induce apoptosis law et al., ; yuan et al., ; khan et al., ) . again, for a and b, the studies were performed in only one or two cell lines. the overexpression of the a protein in vero e (law et al., ) and the overexpression of the b protein in both cos- and vero e cells induce apoptosis khan et al., ) . the overexpression of orf b in vero e cells also induces necrosis (khan et al., ) . a more extensive range of cell lines, including hela, hepg , a , t, cos- and vero e , was used to demonstrate that the overexpression of a can induce apoptosis in cell lines derived from different organs, including lung, kidney and liver . the mechanisms for induction of apoptosis by these sars-cov proteins are unclear, although in some cases, it could be related to their abilities to interfere with cellular functions, such as blocking cell cycle progression, altering membrane permeability, activating signal transduction pathways, upregulating transcription factors and other regulatory genes (table ). these could lead to an imbalance in cellular homeostasis and, consequently, the induction of cell death. the cellular localizations of these sars-cov proteins have also been determined experimentally, and it is likely that they exert their pro-apoptotic effects by interacting with host proteins in these cellular compartments (fig. ) . for example, the sars-cov e protein has the ability to modulate the membrane permeability of mammalian cells (liao et al., ) ion channels in planar lipid bilayers (wilson et al., ) . in addition, yang et al. ( ) showed that the induction of apoptosis by e can be inhibited by the overexpression of bcl-xl, which is a pro-survival member of the bcl- family. as bcl-xl is known to be a critical inhibitor of mitochondrial damage following apoptotic stimuli (dejean et al., ) , it is plausible that e induces apoptosis by perturbing the mitochondrial permeability. however, this has not been demonstrated experimentally. the e proteins of other three coronaviruses, mouse hepatitis virus (mhv), ibv and human coronavirus- e (hcov- e), can also form ion channels in lipid bilayers (wilson et al., ) . interestingly, the mhv e protein has also been shown to induce apoptosis and alter membrane permeability (madan et al., ) . the overexpression of the sars-cov n protein could upregulate the c-jun n-terminal kinase (jnk) and mitogen-activated protein kinase (p mapk) pathways and downregulate the expression levels of extracellularsignal-regulated kinase (erk), phospho-akt and bcl- (surjit et al., ) . further investigations revealed that it could inhibit the activity of cyclin-cyclin-dependent kinase complex and block s-phase progression (surjit et al., ) . the n protein could also activate the transcription factors, nuclear factor kappa b (nf-kb) and activator protein (ap- ) (he et al., ; liao et al., ) . as these transcription factors regulate a wide variety of cellular processes, including cell proliferation, differentiation and apoptosis (hess et al., ; perkins and gilmore, ; tergaonkar, ) , their activation may be linked to the apoptosis-inducing properties of n. for example, the n protein has been shown to upregulate the expression of cyclooxygenase- (cox- ), probably through the activation of nf-kb (yan et al., ) . the s protein can also upregulate cox- expression (liu et al., a) . similar to the e protein, the sars-cov a protein has also been shown to form ion channel in xenopus oocytes (lu et al., ) . the overexpression of the protein b, which was found in both the nucleus and mitochondria, induced cell cycle arrest at the g /g phase . the a protein has been shown to block cell cycle progression at g /g phase by reducing the expression of cyclin d and phosphorylation of retinoblastoma protein (yuan et al., b) . another study showed that the overexpression of a inhibited cellular protein synthesis and induced the phosphorylation and activation of p mapk (kopecky-bromberg et al., ) . the a and a proteins could also activate nf-kb and jnk, leading to the enhancement of il- and rantes production (kanzawa et al., ) . recently, we also demonstrated that the a protein interacts with the pro-survival protein, bcl-x l, and the overexpression of bcl-xl prevents a-induced apoptosis (tan et al., ) . a good correlation between the abilities of a deletion mutants to induce apoptosis and to interact with bcl-x l was observed, suggesting that a triggers apoptosis by interfering directly with the pro-survival function of bcl-xl. mouse hepatitis virus is the one of the most wellcharacterized coronaviruses in terms of its pathogenesis and molecular biology. in particular, a wealth of information is available for the neurotropic john howard mueller (jhm) and the dual hepato-and neurotropic a strains and their effects on the central nervous system (see recent reviews by perlman and dandekar, ; bergmann et al., ) . unlike mhv, infection of young mice ( - weeks) with sars-cov did not result in morbidity or mortality associated with infections in human despite the high level of viral replication in the upper and lower respiratory tracts (glass et al., ; subbarao et al., ; wentworth et al., ) . as such, the regulation of cell death during sars-cov infection has been studied mainly in cell culture systems. here, we shall compare the molecular aspects for the regulation of cell death during infection of immortal cell lines by mhv-jhm, mhv-a and sars coronaviruses. rat oligodendrocytes, which were obtained from the cg- cell line after differentiation in the presence of a low concentration of serum, underwent caspase-dependent apoptosis following infection by mhv-jhm . just as the induction of apoptosis by sars-cov could be inhibited by caspase inhibitors or the overexpression of the pro-survival protein, bcl- (bordi et al., ) , the mhv-induced apoptosis could be inhibited by the caspase- inhibitor and the overexpression of bcl- and bcl-x l, indicating that the mitochondrial pathway is involved further downstream (liu and zhang, ; liu et al., b) . in cl- , a fibroblast cell line, caspasedependent apoptosis was also observed after infection with both mhv-jhm and mhv-a . it was further demonstrated that mhv-a infection of cl- cells activated caspase- , which in turn cleaved bid, a bh -domain pro-apoptotic member of the bcl- family (chen and makino, ) . the resulting tbid p fragment was translocated to the mitochondria, where it induced mitochondrial damage and activation of the caspase cascades. in contrast to these two cell lines, no apoptosis was observed in mhv-a -or mhv-jhminfected dbt cells (a mouse astroytoma cell line) although the cells showed extensive cell fusion and detachment from the plates . curiously, overexpression of the mhv e protein caused apoptosis in dbt cells but not in cl- cells. the reason for the contrasting responses of these cell lines to mhv infection or expression of the e protein remains to be determined. similarly, the overexpression of the e protein of sars-cov can induce apoptosis, and this can be blocked by the bcl-xl protein . as illustrated for sars-cov and mhv, the expression of a single viral protein in immortal cell lines could be sufficient to induce cell death. however, the viral protein may not be an important cell-death regulatory factor during infection. for example, the overexpression of the sars-cov a protein in vero cells resulted in apoptosis, but a mutant virus without the a/ b gene still induced extensive cytopathic effects in vero cells, suggesting that a does not contribute significantly to viral-induced cell death, at least in this cell culture system (yount et al., ) . also, while the overexpression of the mhv e protein induced apoptosis in dbt cells, the expression of e during mhv infection of dbt cells was not sufficient to induce apoptosis . other mechanisms may be more important for regulating apoptosis during infection and this is described in the next section. besides expressing viral proteins that have the abilities to induce apoptosis during infection, viruses can use many other intrinsic and extrinsic mechanisms to modulate cell death in the host cells (see reviews by roulston et al., ; barber, ; hay and kannourakis, ) . here, we further described two other mechanisms that have been documented to be involved in apoptosis induction during coronaviral infection, namely induction of apoptosis via the secretion of soluble factors from neighbouring infected cells (i.e. bystander effects) and fusion of the viral envelope with cellular membranes. numerous coronaviruses [feline infectious peritonitis virus (fipv), oc , e, transmissible porcine gastroenteritis virus (tgev)] have been shown to induce apoptosis in non-infected cells indirectly via the release of soluble cell-death mediators from neighbouring infected cells. in these cases, although viral replication in the apoptotic cells is not required per se, viral replication has to take place in the nearby infected cells. for example, infection of cats with a highly virulent strain of fipv also caused apoptosis in a large number of lymphocytes (haagmans et al., ) . however, apoptosis did not result directly from infection, as many of apoptotic cells were not fipv-antigen positive. thus, apoptosis in fipvinfected cats is occurring via an indirect mechanism. similarly for two human coronaviruses, it is believed that the high level of cytokine secreted from oc -infected mrc- cells or e-infected monocytes/macrophages may be partially responsible for the induction of apoptosis (collins, ; ) . caspase-dependent apoptosis was induced in different cell lines infected with tgev (eleouet et al., ; sirinarumitr et al., ) . again, many of the apoptotic cells were bystander cells as they were not infected by tgev (eleouet et al., ) . on the other hand, no evidence of apoptosis was observed in the intestinal tissues of tgev-infected piglets, suggesting that there may be some host factors that can prevent tgevinduced apoptosis (kim et al., ) . for mhv infection, release of soluble factors has also been shown to be crucial for the induction of demyelination (see a recent review by perlman and dandekar, ) . as for sars-cov-induced apoptosis in vero cells, viral replication is required, but whether apoptosis induction is direct or not has not been established (ren et al., ) . however, there is clear evidence for increased production of certain cytokines and chemokines during sars-cov infection (see recent reviews by cameron et al., ; chen and subbarao, ) , and this could result in apoptosis via a bystander effect. another mechanism that is used by mhv to induce apoptosis in rat oligodendrocytes is dependent on the fusion of the viral envelope with cellular membranes, which led to the activation of the fas signalling pathway (liu and zhang, ) . in this case, uv-inactivated mhv, which is no longer replicative but retains the ability to bind cell receptors and enter the cell, could trigger apoptosis. sars-cov does not appear to induce cell death in this manner, as uv-inactivated sars-cov does not induce apoptosis (ren et al., ) . since the identification of the sars-cov in the year , extensive research on the sars-cov has yielded significant understanding of this newly emerged virus. in terms of cell-death regulation during sars-cov, the occurrence of cell death during infection both in vitro and in vivo has been established, and numerous viral factors have been suggested to contribute to the regulation of cell death in infected cells. although apoptosis and necrosis have been observed in different tissues obtained from sars patients, the ability of sars-cov to induce apoptosis was demonstrated in only one cell line, vero e , and was not extensively studied in animal models. similarly, as summarized in table , many of these studies have investigated the effects of the expression of individual viral proteins on cell death regulation (and other cellular pathways) in a limited number of cell lines; hence it is not clear whether each viral protein can induce cell death in all types of cells. it has also not been determined whether the expressions of these viral proteins are high enough during sars-cov infection, and whether they function in the presence of other viral factors present during infection. given that analysis of host gene transcriptions has suggested that there are significant differences in cellular responses to cell death during infection by coronaviruses sars-cov infection in different cell typs, it is necessary to carry out future investigations in numerous cell lines that support sars-cov replication. clearly, these gaps in our knowledge will be addressed in future studies using infectious clones of sars-cov (yount et al., ; almazan et al., ) and animal models like aged balb/c mice, which, unlike young mice, developed some histopathological damages upon sars-cov infection . in order to understand the regulation of cell death during sars-cov infection, we urgently need to address whether induction of cell death occurs indirectly via bystander effects or directly via expression of viral proteins, or both. for the latter, there is currently no link between the effects of viral proteins on cellular pathways and the induction of apoptosis. to delineate the precise pathways involved, more experiments, like rna interference and mutagenesis studies, are required to establish structure-function relationships. when compared with the other coronaviruses that have been studied for many years, future advancement in understanding the sars-cov may take considerable more time and effort to achieve because of the lack of a single animal model that reproduces all aspects of the human disease (subbarao and roberts, ) and requirement for infection studies to be performed in biosafety level or laboratories. infections of host cells by sars-cov and other coronaviruses have been reported to cause apoptosis. necrosis has also been observed for some coronaviruses. the diverse responses to infection revealed the complex manner by which coronaviruses modulate cell death. furthermore, infection of different cell types by the same virus could also activate distinct cell-death pathways, reflecting the intricate interaction between virus and cell host factors. in order to delineate the contributions of the different viral proteins and viral-host interactions to the regulation of cell death, the correlation between the expression of individual viral proteins and the extent of cell death during infection needs to be established. the availabilities of full-length infectious clones of several coronaviruses and robust animal models provide the essential tools for these future studies. when combined with the technologies to create transgenic or knockout mice and small interfering rna methodologies for specific gene knockdown, such research endeavour will eventually lead to a better understanding of intricate interplay between virus and host. a recent study showed that sars-cov without gene a and b is not as efficient as wild-type virus in inducing dna fragmentation, implying that a and/or b contribute to virus-induced apoptosis in cell culture. schaecher, s.r., touchette, e., schriewer, j., buller, r.m., and pekosz, a. 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( ) analysis of deaths during the severe acute respiratory syndrome (sars) epidemic in singapore: challenges in determining a sars diagnosis. arch pathol lab med : - . chow, k.y., yeung, y.s., hon, c.c., zeng, f., law, k.m., and leung, f.c. ( ) cell death during infection by coronaviruses key: cord- -qlzhtxs authors: goryachev, a.n.; kalantarov, s.a.; severova, a.g.; goryacheva, a.s. title: potential opportunity of antisense therapy of covid- on an in vitro model date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: qlzhtxs data on potential effectiveness and prospects of treatment of new coronavirus infection of covid- caused by virus sars-cov- with the help of antisense oligonucleotides acting against rna of virus on an in vitro model are given. the ability of antisense oligonucleotides to suppress viral replication in diseases caused by coronaviruses using the example of sars and mers is shown. the identity of the initial regulatory section of rna of various coronaviruses was found within - nucleotides from the ’-end, which allows using antisense suppression of this rna fragment. a new rna fragment of the virus present in all samples of coronovirus sars-cov- has been identified, the suppression of which with the help of an antisense oligonucleotide can be effective in the treatment of covid- . the study of the synthesized antisense oligonucleotide ’ - agccgagtgacagcc acacag, complementary to the selected virus rna sequence, was carried out. the low toxicity of the preparations of this group in the cell culture study and the ability to reduce viral load at high doses according to real time-pcr data are shown. the cytopathogenic dose exceeds mg / ml. at a dosage of mg / ml, viral replication is reduced by - times. conclusions are made about the prospects of this direction and the feasibility of using the inhalation way of drug administration into the body. antisense oligonucleotides consists in administering to the body of the patient a drug containing single-strand dna chains which is complementary to any site of single-strand dna or rna, for example, virus rna. complementary binding of dna of the preparation and rna of the virus leads to impossibility of transcription and translation of viral rna and cutting of blocked section of rna of the virus by rnase h. this stops synthesis of new viral particles and prevents intracellular propagation of the virus [ ] . several antiviral antisense drugs have been marketed, for example fomivirsen ® (vitraven ® ) -a drug against cytomegalovirus (novartis), miravirsen ® -a drug for the treatment of hepatitis c (santaris pharma). currently, more than drugs for various diseases based on the use of antisense oligonucleotides are undergoing clinical trials in various phases. antisense therapy is a developing strategy for the specific treatment of new and socially significant diseases. the principle of rna inhibition has previously been studied in vitro to inhibit replication of highly pathogenic rna-viruses [ , ] . thus, given the previous experience of antisense oligonucleotide therapy, it can be assumed that this strategy can be applied as an antiviral drug by binding and cleavage of rna sars-cov- . considering that sars-cov- is an rna virus that does not integrate into the host genome, the strategy of using antisense oligonucleotides, according to some authors, can give effective results [ , ] . prerequisites for the use of antisense therapy in the treatment of covid exist. coronavirus infections have previously caused outbreaks of epidemics. in particular, the outbreak of sars (severe acute respiratory syndrome) in china in - was caused by coronavirus sars-cov, the outbreak of middle eastern respiratory syndrome (mers) was also caused by coronavirus [ , ] . after the sars epidemic, a number of authors conducted studies on the effect of antisense therapy on the suppression of coronavirus growth in the tissues under investigation [ , , ] . in work ( ), the authors found among several sequences the most effective viral transcription inhibitors, suppressing the reproduction of coronavirus in cells and preventing infection of other cells. these are antisense blockers complementary to the initial nucleotides of viral rna -the so-called regulatory transcription sequences of trs (in the terminology of the authors) of the coronavirus strain sars-cov-tor (genbank ay ). the nucleotide sequence of the antisense preparations examined is shown in table : table antisense drugs investigated in work ( ) name of the antisense drugs nucleotide sequence ( ` - `) №№ numbers of blocked viral rna sites of sars-cov -tor trs gttcg tttag agaac agatc - trs taaag ttcgt ttaga gaacag - these sequences are complementary to the following viral nucleic acid sequences (table ) : table genetic target sequences of the coronavirus genome for trs and trs drugs name nucleotide sequence of virus rna ( '- ') site trs gatctgttctctaaacgaac site trs ctgttctctaaacgaacttta * -the standard is to use the notation "t" for uracil in rna the authors used morpholine side chain modification to prevent destruction of antisense oligonucleotide and conjugation with arginine polypeptide to improve penetration into infected cells. by comparison of these nucleotide sequences (table ) to the sequence of a genome of the sars-cov- virus (covid- ) the existence of these sequences almost in all strains of a virus allocated at patients during a pandemic of - was revealed (appendix , the nucleotide sequences are highlighted in red color and a frame). blast analysis (https://blast.ncbi.nlm.nih.gov/blast.cgi ) showed % coincidence of the rna sequence in all coronavirus subtypes, which suggests a high conservatism of this region of the coronavirus genome. this makes it possible to use for the treatment of coronavirus infection those antisense oligonucleotide sequences trs and trs , which were studied in by the collective [ ]. however, starting from mid-march , changes have been observed in the virus strains associated with the loss of sites from the ` end. in particular, the mt usa: wa - - virus sample did not have the regions designated as trs and trs . in this regard, the study of an antisense oligonucleotide complementary to the site of the conserved oligonucleotide sequence of the genome of the sars-cov- virus present in all strains, which was the goal of the present study, seems relevant. in accordance with the purpose of the study, the following tasks were set: -to select the nucleotide sequence of the virus that is supposed to be inhibited, -to carry out the synthesis of oligonucleotide, -to determine cytotoxicity and antiviral activity in an in vitro experiment on cell culture. . selection of nucleotide sequence. the nucleotide sequence that was intended to block in the sars-cov- virus was selected next to the trs and trs genes at a distance of four nucleotides from the final region ( '-end) of the trs gene. this choice was due to the fact that this sequence enters the site regulating transcription (transcription regulatory site, trs) and disabling this sequence will also lead to the impossibility of transcription by analogy with inhibition of trs and trs sites in work [ ] . blast analysis on the genbank (ncbi) showed presence of this sequence at genomes of all sequenced samples sars-cov- . this sequence has the form: `-ctg tgt ggc tgt cac tcg gct in the investigated nucleotide sequences of coronaviruses in appendix , this sequence is highlighted in green. the complementary sequence of the antisense oligonucleotide preparation for treatment has the form: `-agc cga gtg aca gcc aca cag the choice of the nucleotide sequence of the virus for blocking by the antisense preparation was also dictated by the minimal ability to form nucleotide hairpins that prevent hybridization of the rna region of the virus and the antisense preparation. when calculating this sequence on an oligocalculator [ ] , it was found that over a period of nucleotides from the '-end of the viral rna sequence, nucleotides can theoretically form - hairpins, from which the sequence we study can be involved in hairpins, while the sequences trs and trs can be fragments of and nucleotide hairpins, respectively. thus, according to theoretical calculations, we assumed a more specific nature of the selected nucleotide sequence for blocking transcription and translation of the viral genome than previously studied in the work ( ). . synthesis of the drug. the synthesis of an antisense oligonucleotide with phosphorothioate protection of the side sugar-phosphate chain '-agccgagtgacagccacacag was commissioned by genterra, moscow (https://www.genterra.ru/synth.html ). synthesis (medium-scale dna) was carried out with phosphorothioate protection of the phosphate group between all nucleotides with purification of reverse-phase hplc (certificate for synthetic oligonucleotides no. from / / ). the total amount of -membered oligonucleotide synthesized was . mg ( . μmol). the choice of phosphorothioate protection of an oligonucleotide to prevent nuclease degradation of an antisense oligonucleotide is due to the low toxicity of phosphorothioate modifications, commercial availability, the possibility of synthesizing large quantities in routine automatic synthesis on dna synthesizers. the study of toxicity and antiviral activity was carried out by order at the test center for quality control of immunobiological medicines of "national research center for epidemiology and microbiology named after n.f. gamalea "of the ministry of health of russia (study no. / of / / ). the study included the study of the cytotoxic effect of the antisense drug, the study of the antiviral activity of the drug during the therapeutic regimen of drug administration, the detection of sars-cov- rna by real-time pcr. in the experimental work, a transplantable cell line of the kidney of the african green monkey (chlorocebus aethiops) vero-e was used. cell cultivation was carried out in a cell growth medium supplemented with fetal bovine serum (fbs) (final concentration %). the studies used a pandemic strain of human coronavirus sars-cov- "gk / " passage , with an infectious activity of tcid /ml (tissue cytopathogenic doses) for vero e cells from the state collection of viruses of «national research center for epidemiology and microbiology named after n.f. gamalea". the virus was cultured in a vero e cell culture for hours at ° c in a % co atmosphere. infectious activity was determined according to the methods recommended by who. the cytotoxic action of the preparation in vero Е cell culture was determined using -well culture flat-bottomed plates in which vero Е cells were placed at , cells/well in a volume of μl of freshly prepared complete medium. cultivation was carried out by hours at a temperature of °c in the atmosphere of % of co . after incubating the cells with the preparations for hours at a temperature of o c in an atmosphere of % СО , the condition of the cell monolayer was visually evaluated. the culture medium was then removed from the plates and μl of reaction medium and μl of mts ( -( , - solution were added to each well to the cell culture monolayer. after incubation for hours at o c, the results were taken into account on the biorad automatic reader at a wavelength of nm. reference filter was - nm. the concentration of preparations reducing the optical density at nm by % compared to the control of cells was taken as % of the cytotoxic dose (cc ). the experiment to assess the viability of cells in the antiviral efficacy test was carried out in the range of drug concentrations that are not toxic to cells (i.e., lower than the detected cc value). the antiviral activity of the sample was assessed visually under a microscope hours after infection by inhibition of the cytopathic effect of the virus in a vero e cell culture. the result was assessed by Δlg max -the maximum decrease in the value of the infectious viral dose in the experiment in comparison with the control, expressed in decimal logarithms. the study of the antiviral activity of the antisense oligonucleotide substance in the vero e cell culture was carried out with a choice of concentrations based on the results of the cytotoxicity study. working solutions of the test drug were prepared with concentrations of . mg/ml; . mg/ml; . mg/ml and . mg/ml, respectively. the vero e cells used in the study were grown in well culture plates in a volume of μl full for hours at °c in an atmosphere of % co . seed dose - , cells / well. . μl of solutions of the test drug were pipetted from the dilution plates of the test drug to the test plates with cells. each point was tested in parallel wells. the preparations corresponding to the dilution scheme were added to the control wells without virus (to assess the potential cytotoxic effect and further take into account the study results). in the wells of the control cells, a medium was added for staging the reaction. the preparation of dilutions of the viral suspension for the study of antiviral activity was carried out by adding a suspension of sars-cov- , passage , with an infectious activity of tcid / ml for vero e cells to the plates with a monolayer of vero e cell culture: - the suspension was diluted by sequential transfer in test tubes with the required amount of the reaction medium - µl of the reaction medium and µl of the viral suspension. determination of viral production by cytopathic action was carried out on the basis of analysis of cell viability using microscopy, in order to visually determine the boundaries of viral cell damage, as well as to control the toxicity of doses of substances. the assessment of antiviral activity of the drug in addition to cytopathic action was also taken into account by reducing the infectious titer of the virus in the culture of vero cells e according to pcr rna sars-cov- , determined by the threshold of the number of reaction cycles (cycle treshold, ct) in various dilutions of the study drug. the study of sars-cov- rna by pcr was carried out by taking μl of the supernatant from wells with drug dilutions, isolating rna in parallel with positive and negative controls. the result of the study was the conclusion about the presence / absence of sars-cov- rna in the culture liquid when exposed to the drug: the presence of sars-cov- rna (ct value more than ), the absence of sars-cov- rna (ct value is absent). evaluation of the cytotoxicity of the drug at various concentrations was determined by incubating the drug with vero e cells for hours using the mts dye and visual assessment of the cell monolayer. based on the data obtained in the study of the cytotoxic effect of the test substance using mts in the culture of vero e cells, an analytical curve was constructed, from which the cc was determined. determination of the cytotoxicity of the antisense oligonucleotide substance by visual assessment of the state of the monolayer of the vero e cell culture under an inverted microscope did not reveal significant changes in the morphology of cells at a substance concentration of mg/ml and below after hours of incubation of the preparation with cells (table ) . determination of the antiviral efficacy of the antisense oligonucleotide according to the treatment scheme (administration of the drug hours after infection) was taken into account by the decrease in the infectious titer of the virus in the culture of vero e cells by the cytopathic effect. according to the results of the study, it was found that the drug did not inhibit the replication of the sars-cov- virus in the vero e cell culture at the tested concentrations. the results of the study of the antiviral activity of the drug by pcr are presented in table. . it can be seen from the presented data that there are no statistically significant differences between the ct value of the virus control group and the threshold number of cycles in the samples with the addition of the drug. the only difference is the ct value of the group with the addition of the drug at a dosage of . mg/ml, where the ct value is . compared to the virus control group ( . - . ). in the course of research, it was found that the antisense oligonucleotide is low toxic to the culture of vero e cells. the % cytotoxic dose of cc was greater than . mg/ml (exact cytotoxic dose values not determined). at the same time, the results of visual determination of cytotoxicity of the preparations (cc ) were comparable to the results of determination of СС using the vital dye mts. thus, this preparation is low toxic and safe for use. in the course of research, it was found that the antisense oligonucleotide is low toxic to the culture of vero e cells. the % cytotoxic dose of cc was greater than . mg/ml (exact cytotoxic dose values not determined). at the same time, the results of visual determination of cytotoxicity of the preparations (cc ) were comparable to the results of determination of СС using the vital dye mts. thus, this preparation is low toxic and safe for use. as a result of the study of the effectiveness of antisense oligonucleotide in in vitro experiments against sars-cov- , no statistically significant antiviral effect was found in the therapeutic regimen of the drug addition, because according to ( ) the minimum effective virus inhibiting concentration is the concentration of the drug reducing the virus titer by at least . lg. at the same time, as a result of studying the rna content of the virus at various dosages, it was found that with a dosage of the preparation of . - . mg/ml, the parameter ct is the number of amplification reaction cycles (doubling of viral rna), which is necessary to achieve a fluorescent signal is . - . doubling cycles. control ct values in the test with infected cells without the addition of antisense oligonucleotide were . - . values. at a dosage of the same preparation of mg/ml, the ct value was . . this means that at a dosage of mg/ml to achieve a fluorescent signal equivalent to the control group, it was necessary to increase the number of amplification cycles by an average of . - . cycles. this indicates that the dosage of the preparation in mg/ml did not inhibit the full reproduction of the virus, but significantly reduced viral rna replication. the reduction of virus replication based on the calculation of additional amplification cycles [ ] was a range of . - . times ( . - . ). that is, at a dosage of mg/ml of the preparation, the viral load of cells can be reduced by . - times. this value cannot be considered statistically significant, but there is a tendency to reduce the viral load, which may also be effective in antiviral therapy. literature data and data obtained in the experiment indicate that the search for antiviral drugs among groups of antisense oligonucleotides against a new coronavirus infection covid- is a promising direction. a possible way to enhance the antiviral effect can be the use of conjugates of oligonucleotides with other ligands or the use of liposomal forms of drug administration to improve drug penetration into cells. for preclinical and clinical studies of antiviral activity, as well as for therapeutic and prophylactic measures, phosphorothioate protection can be used, as the simplest and cheapest in synthesis. other protected group methods are possible though. considering that the virus spreads by airborne dust and airborne droplets, and affects the epithelium of the respiratory tract and lungs, inhalation of a solution of drugs through a nebulizer can be a promising and convenient method of administration. inhalation of the drug solution through a nebulizer is very simple, does not require sterilization, it reaches the epithelium of the respiratory system in a targeted manner, and is possible even in very severe patients. when antisense oligonucleotides are administered by inhalation, the penetration into the systemic circulation is less than % [ ]. the estimated human dosage is calculated based on the following considerations: the sars-cov- (covid- ) virus is a coronavirus infection that affects the epithelium of the respiratory tract and lungs. theoretically, any cell can be infected, in which up to thousand viral particles can multiply, each of which can infect another cell [ ]. the total area of the lungs and respiratory tract is, on average, m ( mm ). the surface density of alveolocytes per mm is approximately thousand cells ( ). thus, the total number of cells in the lungs and respiratory tract is cells. if we assume that all cells are infected ( , viral particles) and drug molecule is needed for each viral rna, the total number of drug molecules per dose per adult is , or nmol ( μg). a review of antisense oligonucleotides showed that the toxicity of this group of drugs is represented by two types. the first of them -hybridization-dependent toxicity -due to the specific sequence of the oligonucleotide and possible crosslinking with rna, which is not a drug target due to complete or partial coincidence of the nucleotide sequence with the target, as well as possible aptamer binding to proteins [ ] . overcoming this type of toxicity is possible through the proper selection of the target rna, using careful bioinformatics analysis to identify a target with perfect structural match or a small number of mismatched bases. this analysis is performed in the preparatory phase by blast analysis of the blocked sequence with sequences of other genes published in genbank (ncbi). the second type of toxicity is hybridization-independent (nonspecific) toxicity, due to the chemical properties of oligonucleotides interacting with proteins and their decay products. this type of toxicity does not depend on the nucleotide sequence, only on the chemical modification of the sugar-phosphate bridge. from this provision, it follows that if the nucleotide sequence of the antisense preparation is correctly selected for the nucleotide sequence of the virus (coronavirus, influenza virus and other rna viruses), there is no possible coincidence with other genes (according to blast analysis), then toxicity and associated with it, side effects / contraindications will depend only on the chemical modification of the antisense drug, regardless of the nucleotide sequence and the type of inhibited virus. this gives the broadest prospects for the creation of antiviral drugs, not only for coronaviruses, but also for other rna viruses, such as influenza. in this case, it will be sufficient to determine in advance the nonspecific toxicity of the oligonucleotide with the selected protection of the sugar-phosphate backbone. when sequencing the genome of the desired virus, it will be possible to use antisense oligonucleotides as medicinal antiviral agents with accelerated toxicity testing as a kind of off-lable drugs. this, among other things, will provide an opportunity to quickly respond to the threat of the next epidemic as a result of the spontaneous penetration of the virus into the human population, or with the targeted use of the engineered chimeric virus as a weapon or a means of terrorist attack. . a study of literature data has shown the promise of using antisense oligonucleotides in the treatment of viral diseases and, in particular, caused by coronaviruses, such as sars-cov and mers. . oligonucleotides previously studied for sars ( ) are complementary to the nucleotide sequences of the viral rna of the new coronavirus infection covid- in almost all samples and, theoretically, can be considered as drugs for the treatment of the new coronavirus infection covid- . investigation of an antisense oligonucleotide with phosphorothioate protection `-agccgagtgacagccacacag, complementary to the region of viral rna near the `-end, showed extremely low toxicity. the cc dose in studies on vero e cell culture was more than mg/ml. . the study of the antiviral activity of antisense oligonucleotide on vero cell culture e according to real-time pcr showed that at a dosage of mg/ml, the reduction in viral sars-cov- endothelial infection causes covid- chilblains: histopathological, immunohistochemical and ultrastructural study of seven paediatric cases sars and mers: recent insights into emerging coronaviruses Руководство по проведению доклинических исследований лекарственных средств oligocalc: an online oligonucleotide properties calculator examination of antisense rna and oligodeoxynucleotides as potential inhibitors of avian leukosis virus replication in rp cells the real-time polymerase chain reaction// molecular aspects of medicine development of therapeutics for treatment of ebola virus infection antisense oligonucleotides target a nearly invariant structural element from the sars-cov- genome and drive rna degradation antisense morpholino-oligomers directed against the ` end of the genome inhibit coronavirus proliferation and growth// journal of virology antiviral effects of antisense morpholino oligomers in murine coronavirus infection models// journal of virology sars and other coronaviruses as causes of pneumonia key: cord- -qqhivgkq authors: chang-liao, wan-ping; lee, an; chiu, yu-han; chang, hui-wen; liu, je-ruei title: isolation of a leuconostoc mesenteroides strain with anti-porcine epidemic diarrhea virus activities from kefir grains date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: qqhivgkq swine grown under commercial conditions are vulnerable to environmental exposure to several viruses, which may cause infectious diseases and spread easily and rapidly, resulting in significant economic losses in animal husbandry. previous studies have suggested that probiotics seem to be a new and promising alternative to vaccinations to protect animals against potential viral infections. in this study, we used the vero cell culture model of infection to study porcine epidemic diarrhea virus (pedv). we screened lactic acid bacteria (lab) with anti-pedv potential from kefir grains, which are starter cultures used to ferment milk into kefir. twenty-nine lab strains were isolated and identified as enterococcus durans, lactobacillus kefiri, lactococcus lactis, and leuconostoc mesenteroides, according to s ribosomal rna (rrna) and rpoa gene sequence analyses. the anti-pedv activities of the lab intracellular extracts were compared, and the intracellular extracts of ln. mesenteroides showed higher anti-pedv activities than that of the other species. among the ln. mesenteroides strains, a strain designated ypk showed a higher growth rate than that of the other strains and was further evaluated for its anti-pedv activity. the results showed that the intracellular extracts of ln. mesenteroides ypk possessed in vitro prophylactic, therapeutic, and direct-inhibitory effects against pedv in the vero cell model. the expression levels of type interferon (ifn)-dependent genes, including myxovirus resistance (mx ) and interferon-stimulated gene (isg ), were significantly increased after treatment with intracellular extracts of ln. mesenteroides ypk for h. such expression suggests that the anti-pedv activity of ln. mesenteroides ypk could be attributed to its up-regulatory effect on the expression of mx and isg genes. these results suggested that ln. mesenteroides ypk has the potential to provide some levels of host protection against pedv infections. in order to increase swine and poultry production, it is common to raise animals in high-density populations. swine grown under commercial conditions are vulnerable to environmental exposure to several viruses. some viruses may cause infectious diseases, which are spread easily and rapidly cause significant economic losses in animal husbandry. vaccination is one of the most efficient strategies to prevent viral diseases and control infections and is the current industry standard. efficacious vaccines have been developed and applied successfully for the prevention of several infectious viral diseases in swine, such as porcine parvovirus (ppv), foot-and-mouth disease virus (fmdv), porcine circovirus type (pcv ), and transmissible gastroenteritis virus (tgev; meng, ; gerdts and zakhartchouk, ) . however, some of the vaccination methods require the operator to handle each animal, induce the stress on the animal, and are time-consuming and costly (marangon and busani, ) . although efficacious vaccines are available to reduce the impact of the infectious viruses mentioned above, unfortunately, substantial challenges remain in obtaining safe and efficacious vaccines for a variety of newly emerging and re-emerging viruses, such as african swine fever virus and porcine epidemic diarrhea virus (pedv; lee, ) . pedv is a member of the genus alphacoronavirus in the family coronaviridae of the order nidovirales and has emerged as a significant pathogen causing lethal watery diarrhea, vomiting, and dehydration in nursing piglets. highly pathogenic strains of pedv have mortality rates of - % in neonatal piglets, which has resulted in huge economic losses to the swine industry worldwide (koonpaew et al., ; wang et al., ) . accumulated evidence indicates that pedv encodes defensive mechanisms to evade virus recognition by host pattern recognition receptors (prrs) present on antigen-presenting cells, inhibit interferon (ifn) induction, and antagonize ifn signaling and antiviral effector machinery (hao et al., ; koonpaew et al., ) . pedv has been studied extensively and some vaccines have been developed against pedv, but the efficacy of these vaccines in the field remains questionable (wang et al., ) . probiotics, which are live microorganisms that when administered in adequate amounts confer a health benefit on the host, have long been used as feed additives because of their abilities to normalize gut microbiota, boost the immune system, prevent diarrhea, and improve feed conversion efficiency (fontana et al., ; alonso and guarner, ) . the effect of probiotics is achieved mainly through the intervention on gut microbiota, which increases the levels of beneficial bacteria and decreases the pathogenic populations in the gastrointestinal tract (liu et al., ; yadav and shukla, ) . in addition to the microbiota-modulatory properties, recent studies showed that the immunomodulatory activity of probiotics is another important mechanism of action of probiotics for the inhibition of pathogens (llewellyn and foey, ; azad et al., ) . those immunoregulatory probiotics provide host protection against pathogenic infections by modulating innate and adaptive antiviral immune responses (villena et al., ) . several probiotic strains, most of them belonging to lactobacillus and bifidobacterium genera, have been shown to perform antiviral activities (villena et al., ; arena et al., ) . these antiviral activities could be mediated by the immunomodulatory properties of probiotics because it was observed that administration of probiotics induced the expression of ifn and interferonstimulated genes (isgs), which are crucial components of the ifn responses and play a key role in establishing an antiviral state for virus clearance and restriction of spread (zelaya et al., ; villena et al., ; arena et al., ; eguchi et al., ) . if probiotics have antiviral activity, it seems to be a new and promising alternative to vaccinations to protect animals against potential viral infections (al kassaa et al., ) . kefir is an acidic and mildly alcoholic fermented milk product that is believed to have many beneficial activities, such as hypocholesterolemic activity, antibacterial and antifungal activities, antitumor activity, immunomodulatory activity, and quickening of wound healing (bourrie et al., ) . traditionally, kefir is produced from milk fermented with a mixed microflora confined to a matrix of discrete kefir grains, which are a combination of bacteria and yeasts in a symbiotic matrix (marshall and cole, ) . various bacteria and yeasts have been identified in kefir grains. the bacteria present in kefir grains may include acetobacter, bifidobacterium, lactobacillus, lactococcus, leuconostoc, oenococcus, and streptococcus, while the yeasts present in kefir grains may include candida, kluyveromyces, and pichia (lin et al., ; wang et al., ; bourrie et al., ) . numerous bacterial strains with specific properties, such as hypocholesterolemic effect, antiallergenic effect, immunoregulatory effects, and antipathogenic activities, have been isolated from kefir grains (prado et al., ) . however, to the best of our knowledge, following a thorough review of the relevant literature, there has been no study to isolate bacterial strains with antiviral activities from kefir grains. in the present study, we used the vero cell culture model of infection by pedv to screen lactic acid bacteria (lab) with anti-pedv activities from kefir grains. the anti-pedv activities of the lab strains were evaluated in prophylactic, therapeutic, and direct-inhibitory models. the strains with anti-pedv activities were further studied to define their effects on the expression of type ifn-dependent genes, including '- '-oligoadenylate synthetase (oas ), myxovirus resistance (mx ), and interferon-stimulated gene (isg ) in vero cells. a total of lab strains were isolated from kefir grains according to the method described by lin et al. ( ) . the lab isolates were cultured in de mann, rogosa, sharpe (mrs) broth (oxoid, basingstoke, uk) at °c for h without shaking. the bacterial concentrations were measured by optical density readings at nm or by traditional colony counting methods (vinderola et al., ) . for colony morphological observation, the lab isolates were cultivated on mrs agar plates (oxoid) or blood agar plates frontiers in microbiology | www.frontiersin.org (merck, darmstadt, germany) at °c for h and then observed. for cell morphological observation, the lab isolates were cultured in mrs broth (oxoid) at °c for h without shaking. the bacterial cells were harvested by centrifugation at , g for min at °c, stained with gram stain (sigma-aldrich, st. louis, mo, usa) according to the manufacturer's instructions, and observed under a microscope. unstained bacteria were observed under a phase-contrast microscope. molecular identification of the bacterial isolates was performed by the methods described by weisburg et al. ( ) and naser et al. ( ) . genomic dna of the bacterial isolates was isolated using the dneasy blood & tissue kit (qiagen inc., valencia, ca, usa). the standard s ribosomal rna (rrna) gene primers and rna polymerase α subunit gene rpoa primers were used for pcr to amplify the s rrna gene and rpoa gene sequences of the bacterial isolates, respectively (weisburg et al., ; naser et al., ) . the resultant pcr products were sequenced by an automatic sequencing service provided by genomics biotech inc., (new taipei city, taiwan). the nucleotide sequences of the s rrna and rpoa genes were aligned using the national center for biotechnology information's basic local alignment search tool (blast). for the determination of biochemical characteristics, the carbon source use profiles of the lab isolates were determined using an api ch system (biomerieux, inc., marcy l'etoile, france) according to the manufacturer's instructions. for the determination of physiological characteristics, the lab isolates were cultured at °c, °c, ph . , or in the presence of % ethanol according to the methods described by lin et al. ( ) . before the anti-pedv activity assay, the cytotoxicity of the lab isolates on the african green monkey kidney cell line vero was evaluated according to the method described by mosmann ( ) . the vero c cells were purchased from the bioresource collection and research center of food industry research and development institute (bcrc, hsinchu, taiwan) and were routinely grown at °c in a humidified atmosphere of % air and % co in dulbecco's modified eagle's medium (dmem, gibco, grand island, ny, usa) supplemented with % fetal bovine serum (fbs; moregate biotech., queensland, australia). for evaluation of cytotoxicity, a . -ml aliquot of the -h culture of each bacterial strain was centrifuged at , g for min at °c. the bacteria were collected, washed twice with sterile phosphate-buffered saline (pbs; . m, ph . ), resuspended in . ml of sterile pbs, and sonicated for min with an ultrasonicator (model xl, misonix, farmingdale, ny). the sonicated bacteria were fractioned into intracellular extracts and cell-wall pellet fractions by subsequent centrifugation at , g for min at °c. the intracellular extracts were harvested, and the cytotoxicity on vero cells was determined using the -( , -dimethylthiazol- -yl)- , diphenyltetrazolium bromide (mtt) assay according to the method described by mosmann ( ) . briefly, vero cells were seeded at a density of . × cells/well on a -well plate in μl of dmem. after incubating at °c for h, μl of the bacterial intracellular extracts were added into each well and incubated at °c for another h. after washing twice with sterile pbs, the cells were incubated with μl of mtt ( mg/ml in pbs) at °c for h. after the incubation, the medium was removed, and μl of dimethyl sulfoxide (dmso) were added into each well to dissolve the formazan crystals. the absorbance was measured at nm using a microplate reader (victor , perkinelmer inc., waltham, ma, usa), and percentages of cell metabolic activity were calculated as follows: absorbance of sample % / where the absorbance of sample is the absorbance of cells treated with test sample and the absorbance of control is the absorbance of cells treated with dmso. the pedv taiwan pintung strain was isolated in early from the intestinal homogenate of a -day-old suckling pig in taiwan and adapted to vero cells as previously described by chang et al. ( ) . viral infection and propagation were performed in vero cells according to the method described by chang et al. ( ) . before the anti-pedv activity screening experiments were conducted, the viral titers of pedv were adjusted to fifty-percent tissue culture infective dose (tcid )/ml, and the intracellular extracts of bacterial isolates were prepared as described above. vero cells were seeded at a density of × cells/well on a -well plate in μl of modified postinoculation (pi) medium containing dmem (gibco, grand island, ny, usa) supplemented with tryptose phosphate broth ( . %), yeast extract ( . %), and μg/ml of trypsin. after incubating at °c for h, μl of the bacterial intracellular extracts were added into each well and incubated at °c for another h. after washing the cells twice with pi medium, μl of pi medium containing tcid /ml of the pedv was added into each well and incubated at °c for h. after h of incubation, the supernatants were replaced by fresh pi medium and the cells were maintained at °c for h. after washing twice with sterile pbs ( . m, ph . ), the cell metabolic activity was determined using the mtt assay as described previously. effects of lab intracellular extract, cell-wall fraction, and extracellular supernatant against pedv a . -ml aliquot of the -h culture of each lab strain was centrifuged at , g for min at °c. the resultant extracellular supernatants and bacterial cells were harvested separately. the bacterial cells were washed twice with sterile pbs, resuspended in . ml of sterile pbs, and sonicated for min with an ultrasonicator (model xl, misonix, farmingdale, ny, usa). the sonicated bacterial cells were fractioned into intracellular extracts and cell-wall pellet fractions by subsequent centrifugation at , g for min at °c. the extracellular supernatants, intracellular extracts, and cell-wall fractions of each bacterial strain were harvested, and the anti-pedv activities were evaluated as described above. vero cells were seeded on a -well plate and incubated at °c for h. afterward, the cells were washed with pi medium, and μl of pi medium containing tcid /ml of pedv were added into each well and incubated at °c for h. after h of incubation, the supernatants were replaced by μl of fresh pi medium and μl of the bacterial intracellular extracts. the cells were maintained at °c for h. after washing twice with sterile pbs, the cell metabolic activity was determined by mtt assay as described previously. . glyceraldehyde -phosphate dehydrogenase (gapdh) was chosen as an internal control, and all relative gene expression levels were normalized to gapdh by the comparative c t method. the primers used for the relative quantification are provided in table . the data were analyzed using spss version software (ibm spss, new york, ny, usa). one-way analysis of variance (anova) followed by duncan's multiple range test was used to detect the differences among the means of the different treatment groups, and a value of p less than . was considered significant. student's t-test was used to detect the differences between the treatment and control groups, and a value of p less than . was considered significant. each experiment was conducted in triplicate, and all results were expressed as means ± standard deviations. twenty-nine lab strains were isolated from kefir grains. according to the s rrna and rpoa gene sequence analysis, three isolated strains belong to the species enterococcus durans, isolated strains might belong to the species lactobacillus kefiri, five isolated strains might belong to the species lactococcus lactis, and five isolated strains might belong to the species leuconostoc mesenteroides ( table ). the in vitro prophylactic effects of the lab strains on pedv were evaluated in the vero cell model. vero cells were pretreated with the intracellular extracts of lab for h. after the removal of the bacterial intracellular extracts, the vero cells were infected with pedv. if the intracellular extracts of lab possessed an in vitro prophylactic effect against pedv, the bacterial pretreated vero cells would be infected with less pedv and thus would show higher cell metabolic activity than the un-pretreated cells. the in vitro prophylactic effects of the bacterial intracellular extracts of different lab species against pedv were compared, and human ifn-α b was used as a positive control. vero cells pretreated with ifn-α b prior to pedv infection showed a significantly higher cell metabolic activity and less cytopathic effects than the un-pretreated cells (figures , ) , indicating that ifn-α b possessed an in vitro prophylactic effect against pedv. among the cells pretreated with the intracellular extracts of different lab species, the cells pretreated with the intracellular extracts of ln. mesenteroides showed significantly higher cell metabolic activity than those pretreated with the intracellular extracts of the other lab species (figure ). in addition, vero cells pretreated with the intracellular extracts of ln. mesenteroides also showed less cytopathic effects than the un-pretreated cells (figure ) , indicating that ln. mesenteroides possessed an in vitro prophylactic effect against pedv. the in vitro prophylactic effects of the five strains of ln. mesenteroides isolated from kefir grains on pedv were further compared with each other. as shown in figure , the metabolic activity of vero cells pretreated with the intracellular extracts of ln. mesenteroides, regardless of which strain, were similar to those pretreated with ifn-α b (p > . ) but were significantly higher than the un-pretreated cells (p < . ), indicating that all the ln. mesenteroides strains isolated from kefir grains possessed in vitro prophylactic effects against pedv. in vitro prophylactic effects of ln. mesenteroides ypk intracellular extract, cell-wall fraction, and extracellular supernatant against pedv among the ln. mesenteroides strains, a strain designated ypk showed a higher growth rate than the other strains (data not shown) and was further evaluated for its basis of anti-pedv activity. we compared the in vitro prophylactic effects of intracellular extracts, cell-wall fractions, and extracellular supernatants of ypk against pedv. as shown in figure , the metabolic activity of vero cells pretreated with the intracellular extracts and extracellular supernatants of ypk were similar to each other and were significantly higher than those of un-pretreated cells or those pretreated with the cell-wall fractions of ypk (p < . ), indicating that both of the intracellular extracts and extracellular supernatants of ypk possessed in vitro prophylactic effects against pedv. vero cells were infected with pedv for h, and then the remaining pedv was removed. the pedv-infected cells were treated with the intracellular extracts of ypk or ifn-α b for h, and then the metabolic activities of vero cells were determined. as shown in figure , pedv-infected vero cells treated with the intracellular extracts of ypk for h showed higher cell metabolic activity than the untreated cells or those treated with ifn-α b (p < . ), indicating that the intracellular extracts of the ypk strain possessed an in vitro therapeutic effect against pedv. vero cells were co-incubated with pedv and the intracellular extracts of ypk or ifn-α b for h. after removal of the pedv and intracellular extracts of ypk , the vero cells were incubated for h, and then the metabolic activities of the vero cells were determined. the metabolic activities of the vero cells treated with the intracellular extracts of ypk and pedv simultaneously were significantly higher than those treated with pedv alone or those treated with pedv and ifn-α b (figure ; p < . ). these results suggested that the intracellular extracts of ypk possessed an in vitro direct-inhibitory effect against pedv in vero cells. in order to elucidate the antiviral mechanisms of the ypk strain, the expression levels of type ifn-dependent genes, including oas , mx , and isg , were quantified in vero cells after , , or h of treatment with the intracellular extracts of ypk . as shown in figure , ifn-α b, which served as the positive control, significantly increased the expression levels of oas , mx , and isg genes in vero cells at and h. the intracellular extracts of ypk also significantly increased the expression levels of mx and isg genes but did not affect the expression levels of the oas gene in vero cells at h. however, the expression levels of oas , mx , and isg genes in vero cells did not differ between the untreated and ypk -treated groups at h. according to the s rrna and rpoa gene sequence analysis, ypk exhibited . and . %, respectively, identity with ln. mesenteroides (table ) , and its s rrna and rpoa gene sequences were deposited in the ncbi genbank database under accession number mt and mt , respectively. macroscopic observation showed that ypk exhibited a smooth and grayish white colony morphology and did not possess hemolytic capacity on blood agar plate. the cells of ypk appeared purple after gram staining, indicating the strain ypk was gram positive. microscopic observation showed the cells of ypk were observed as spherical or lenticular forms. analysis on the basis of phenotypic (including gramstain-positive, catalase-negative, nonmotile, and asporogenous) and physiological characteristics (including growth at or °c but not growth at ph . or in % ethanol) indicated that ypk was closely related to species ln. mesenteroides. to further confirm the identification of ypk with the species ln. mesenteroides, the biochemical characteristics of ypk were compared with those of ln. mesenteroides subsp. cremoris atcc , ln. mesenteroides subsp. dextranicum atcc , and ln. mesenteroides subsp. mesenteroides atcc by using the api ch system. analysis of carbon all data are expressed as mean ± sd (n = ). bars marked with a star or double stars mean that they are significantly different from the control (cells treated with pbs) at the or % confidence level, respectively. source utilization profiles indicated that both ypk and ln. mesenteroides subsp. dextranicum atcc grew on six out of carbohydrates, including n-acetyl glucosamine, d-fructose, d-glucose, d-mannose, saccharose, and d-trehalose. distinct variation was observed between ypk and ln. mesenteroides subsp. cremoris atcc in the metabolism of the sugars d-fructose, d-mannose, and d-trehalose. additionally, distinct variation was observed between ypk and ln. mesenteroides subsp. cremoris atcc in the metabolism of the carbohydrates amygdaline, l-arabinose, cellobiose, esculine, d-galactose, β-gentiobiose, d-lactose, maltose, α-methyl-d-glucoside, d-raffinose, ribose, d-turanose, and d-xylose. therefore, the carbon source use characteristics of ypk were similar to those of ln. mesenteroides subsp. dextranicum. according to the results of microscopic observations, biochemical characteristics, and the s rrna and rpoa gene sequence analysis, the features of ypk were consistent with those of ln. mesenteroides subsp. dextranicum, as described in bergey's manual of systematic bacteriology (vos et al., ) . the vero cell line is one of the most commonly used cell lines for pedv isolation and propagation (koonpaew et al., ) . in this study, we used a vero cell culture model to evaluate the in vitro prophylactic effects of lab against pedv. four lab species, including e. durans, lb. kefiri, lc. lactis, and ln. mesenteroides , were isolated from kefir grains, and the in vitro prophylactic effects of the intracellular extracts of these four species against pedv infection in vero cells were compared. among these four lab species, the intracellular extracts of ln. mesenteroides showed a higher in vitro prophylactic effect against pedv than the other species did (figure ) . in addition to the in vitro prophylactic effect, the intracellular extracts of ln. mesenteroides ypk also possessed in vitro therapeutic effect and in vitro direct-inhibitory effects against pedv in vero cells (figures - ) . vero cells have a major deletion in the type ifn gene cluster, which results in ifn deficiency (desmyter et al., ; koonpaew et al., ) . although vero cells do not secrete type ifns when infected by viruses, they still have the type ifn receptors and respond normally to type ifns. therefore, vero cells were widely used to compare virus-mediated ifn antagonism specific to the ifn signaling pathway (simmons et al., ) . in the in vitro prophylactic and therapeutic models, the intracellular extracts of ln. mesenteroides ypk did not directly interact with pedv by physical contact. therefore, the in vitro prophylactic and therapeutic effects of the intracellular extracts of ln. mesenteroides ypk against pedv in vero cells seem not be attributed to the direct interaction of bacterial components or metabolites with virus. since the ifn pathway is crucial in initiating viral resistance, we suggest that the in vitro prophylactic and therapeutic effects of the intracellular extracts of ln. mesenteroides ypk against pedv in vero cells could be attributed to its effect on the ifn signaling pathway in vero cells. stimulation of innate immune responses by probiotics could be one of the mechanisms responsible for the protection provided by probiotics against viral infection. other proposed mechanisms include inhibition of virus adsorption and penetration into cells as a result of direct interaction of probiotics and virus, competition between probiotics and virus for epithelial cell receptors, and secretion of metabolites with antiviral activity (mousavi et al., ; biliavska et al., ) . previous studies have shown that specific probiotic bacteria bind to and inactivate rotaviruses and vesicular stomatitis viruses, which lead to blocking of the virus adsorption on the cell (salminen et al., ; kanauchi et al., ) . besides the direct interaction between probiotics and viruses, specific probiotic bacteria could interact with epithelial and mucosal cells and compete with pathogens for attachment to cell receptors, thereby preventing invasion into the cells by a virus (fernandez-duarte et al., ; mousavi et al., ) . in addition, specific probiotic bacteria could synthesize some antiviral metabolites, such as lactic acid, hydrogen peroxide, or bacteriocins (al kassaa et al., ) . in the present study, the intracellular extracts of ln. mesenteroides ypk possessed an in vitro direct-inhibitory effect against pedv in vero cells (figure ) . future studies will be aimed at identifying the mechanisms of the in vitro direct-inhibitory effect of the intracellular extracts of ln. mesenteroides ypk against pedv in vero cells. numerous mechanisms for the immunomodulatory properties of probiotics have been proposed. some extracellular polysaccharides produced by specific probiotic bacteria possess immunomodulatory activities, which induce an increase in the expression of ifn-α, ifn-β, and the antiviral factors mx and rnase l in porcine intestinal epithelial cells (kanmani et al., ) . beside extracellular polysaccharides, some cellular components, such as dna and bacterial cell-wall components, including peptidoglycan, s-layer proteins, teichoic acids, capsule, and pellicle, as well as other released peptides could modulate the innate antiviral immune response (quinteiro-filho et al., ) . in the present study, pretreatment of vero cells with the extracellular supernatants or intracellular extracts of ln. mesenteroides ypk for h before infection with pedv showed higher cell metabolic activities than those of un-pretreated cells, indicating that both of the intracellular extracts and extracellular supernatants of ln. mesenteroides ypk possessed in vitro prophylactic effects against pedv in vero cells. however, pretreatment of pedv cells with the cell-wall fractions of ln. mesenteroides ypk for h before infection with pedv did not impede pedv replication. according to these observations, we suggested that the immunomodulatory activity of ln. mesenteroides ypk seems not to rely on the structural cell components. future studies should be aimed at assessing the molecular mechanism(s) responsible for the observed effects. type ifns exert their antiviral activities though the induction of hundreds of isgs (lenschow et al., ) . classical isgs responsible for inhibition of viral infection include oas , mx , and isg (schoggins, ) . oas , which belongs to the oas enzyme family, is activated by double-stranded rna binding, catalyzes the formation of ′- ′ oligoadenylates to activate cellular rnase l, which in turn, degrade cellular and viral rna (choi et al., ) . mx is a dynamin-like gtpase that appears to target viral nucleocapsids, resulting in the inhibition of viral rna polymerase activity, effectively blocking both transcription and replication of the virus (schoggins, ) . isg is a small, ubiquitin-like molecule that has numerous antiviral functions, including inhibition of virus release, isgylation of both viral and host proteins, and immunomodulatory cytokinelike properties in its unconjugated form (schoggins, ) . in the present study, we determined the effects of intracellular extracts of ln. mesenteroides ypk on the expression levels of oas , mx , and isg genes in vero cells and found that treatment of vero cells with the intracellular extracts of ln. mesenteroides ypk did not affect the expression levels of the oas gene but significantly increased the expression levels of mx and isg genes h after treatment (figure ) , indicating that the anti-pedv activity of the intracellular extracts of ln. mesenteroides ypk could be attributed to its up-regulatory effect on the expression of mx and isg genes in vero cells. according to the results of microscopic observations, biochemical characteristics, and the s rrna and rpoa gene sequence analysis, ypk was identified as ln. mesenteroides subsp. dextranicum. ln. mesenteroides are commonly associated with foods, such as fermented dairy products (e.g., cheese, yogurt, and kefir), fermented vegetables (e.g., sauerkraut and kimchi), and fermented meats (holland and liu, ) . the long history of safe consumption of ln. mesenteroides in traditional fermented foods has led to the conclusion that it is generally regarded as safe (gras; flórez et al., ) . several ln. mesenteroides strains are reported to have anti-listerial, antiviral, or immunomodulatory activities (seo et al., ; shao et al., ) . since the production of exopolysaccharides and bacteriocins are important properties of ln. mesenteroides, the probiotic characteristics of ln. mesenteroides could be attributed to their production of exopolysaccharides and bacteriocins. several studies demonstrated that some bacteriocins produced by ln. mesenteroides have anti-pathogenic activities (stiles, ; holland and liu, ; arakawa et al., ) , and some exopolysaccharides produced by ln. mesenteroides showed antiviral and immunomodulatory activities (nácher-vázquez et al., ; mahdi et al., ) . in our study, we found that the intracellular extracts of ln. mesenteroides ypk possessed in vitro prophylactic, therapeutic, and direct-inhibitory effects against pedv in vero cells, which occur in part through the up-regulation of mx and isg expression in vero cells. to the best of our knowledge, based on a thorough review of the relevant literature, this scientific report is the first of anti-pedv potential for ln. mesenteroides. future research will be conducted to evaluate the protection efficiency of ln. mesenteroides ypk against pedv infections in piglet infectious challenge models. the lab strain ypk isolated from kefir grains was genotypically and phenotypically characterized as belonging to ln. mesenteroides subsp. dextranicum. ln. mesenteroides subsp. dextranicum ypk displayed in vitro prophylactic, therapeutic, and direct-inhibitory effects against pedv in vero cells via up-regulation of mx and isg expression in vero cells. these findings suggest that ln. mesenteroides subsp. dextranicum ypk has a potential to acts as an antiviral agent for protection against pedv infections. the datasets presented in this study can be found in online repositories. the names of the repository/repositories and accession number(s) can be found in the article/supplementary material. j-rl, w-pc-l, and h-wc contributed to conception and design of the study. w-pc-l, al, and y-hc carried out the experiments and did the data analysis. w-pc-l and j-rl wrote the first draft of the manuscript. all authors contributed to the manuscript revision, read and approved the submitted version. the corresponding author takes primary responsibility for communication with the journal and editorial office during publication. antiviral potential of lactic acid bacteria and their bacteriocins linking the gut microbiota to human health production of a bacteriocin-like inhibitory substance by leuconostoc mesenteroides subsp. dextranicum m isolated from mongolian fermented mare milk, airag immunobiosis and probiosis: 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beneficial effects against rotavirus infection viability of probiotic (bifidobacterium, lactobacillus acidophilus and lactobacillus casei) and nonprobiotic microflora in argentinian fresco cheese bergey's manual of systematic bacteriology investigation of microorganisms involved in biosynthesis of the kefir grain emerging and re-emerging coronaviruses in pigs s ribosomal dna amplification for phylogenetic study recent systems biology approaches for probiotics use in health aspects: a review nasal priming with immunobiotic lactobacillus rhamnosus modulates inflammation-coagulation interactions and reduces influenza virus-associated pulmonary damage this research was conducted using funds partially provided by grants from the ministry of science and technology (grant nos. most - -b- - and most - -b- - ) and academia sinica (grant nos. as-kpq- -itar-td and as-kpq- -itar-td ). key: cord- - b authors: ianevski, aleksandr; yao, rouan; fenstad, mona høysæter; biza, svetlana; zusinaite, eva; reisberg, tuuli; lysvand, hilde; løseth, kirsti; landsem, veslemøy malm; malmring, janne fossum; oksenych, valentyn; erlandsen, sten even; aas, per arne; hagen, lars; pettersen, caroline h.; tenson, tanel; afset, jan egil; nordbø, svein arne; bjørås, magnar; kainov, denis e. title: potential antiviral options against sars-cov- infection date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: b as of june , the number of people infected with severe acute respiratory coronavirus (sars-cov- ) continues to skyrocket, with more than . million cases worldwide. both the world health organization (who) and united nations (un) has highlighted the need for better control of sars-cov- infections. however, developing novel virus-specific vaccines, monoclonal antibodies and antiviral drugs against sars-cov- can be time-consuming and costly. convalescent sera and safe-in-man broad-spectrum antivirals (bsaas) are readily available treatment options. here, we developed a neutralization assay using sars-cov- strain and vero-e cells. we identified the most potent sera from recovered patients for the treatment of sars-cov- -infected patients. we also screened safe-in-man broad-spectrum antivirals against the sars-cov- infection in vero-e cells and identified nelfinavir, salinomycin, amodiaquine, obatoclax, emetine and homoharringtonine. we found that a combination of orally available virus-directed nelfinavir and host-directed amodiaquine exhibited the highest synergy. finally, we developed a website to disseminate the knowledge on available and emerging treatments of covid- . every year, emerging and re-emerging viruses such as sars-cov- , sars-cov, middle east respiratory syndrome coronavirus (mers-cov), zika virus (zikv), ebola virus (ebov), influenza a virus (fluav) and rift valley fever virus (rvfv) surface from natural reservoirs to infect people [ , ] . as of june , the number of people infected with sars-cov- continues to rise, with more than . million cases worldwide. we selected subjects among healthy individuals, in-and outpatients, as well as patients recovered from sars-cov- or endemic coronavirus infections. hospitalization was determined by whether a patient was able to manage symptoms effectively at home, according to local guidelines. icu admittance was evaluated consistently with the who interim guidance on "clinical management of severe acute respiratory infection when covid- is suspected" (who/ -ncov/clinical/ . ). the patients gave their informed consent through the koronastudien.no website. for icu patients, consent for sample collection was received after treatment or from relatives. for children, consent for sample collection was received from their parents. donors were recruited through information at the blood collection centers websites and through national media. nasopharyngeal swabs (nps) and blood samples were collected. all patients were treated in accordance with good clinical practice, following study protocols. the study was approved by the national ethical committee (clinical trial: nct ; rek: ). human telomerase reverse transcriptase-immortalized retinal pigment epithelial (rpe), lung adenocarcinoma a , non-small-cell lung cancer calu- and epithelial colorectal adenocarcinoma caco- cells were grown in dmem-f supplemented with µg/ml streptomycin and u/ml penicillin (pen-strep), mm l-glutamine, % fbs and . % sodium bicarbonate (sigma-aldrich, st. louis, mo, usa). human neural progenitor cells derived from ips cells were generated and maintained as described previously [ ] . human large-cell lung carcinoma nci-h , colon cancer sw , colorectal carcinoma sw and ht cells were grown in rpmi medium supplied with % fbs and pen-strep. human adenocarcinoma alveolar basal epithelial a , human embryonic kidney hek- cells and kidney epithelial cells extracted from an african green monkey (vero-e ) were grown in dmem supplemented with % fbs and pen-strep. the cell lines were maintained at • c with % co . the sars-cov- strains were isolated and propagated in a biological safety level (bsl- ) facility. two hundred microliters of nasopharyngeal swabs (nps) in universal transport medium were diluted times in culture medium (dmem) supplemented with . % bovine serum albumin (bsa), . µg/ml penicillin, µg/ml streptomycin and mm l-glutamine and inoculated into vero-e cells. after days of incubation, the media were collected, and the viruses were passaged once again in vero-e cells. after days, a clear cytopathic effect (cpe) was detected, and the virus culture was harvested. virus concentration was determined by rt-qpcr and plaque assays. viral rna was extracted using the ntnu_mag_v protocol, a modified version of the bomb-protocol [ ] . the eluate ( . or µl) was analyzed by rt-qpcr using a cfx real-time thermal cycler (bio-rad, hercules, ca, usa) as described elsewhere [ ] , with some modifications. one -µl reaction contained µl of qscript xlt one-step rt-qpcr toughmix ( ×) (quanta biosciences, beverly, ma, usa), µl each of the primer and probe with final respective concentrations of . and . µm and µl of molecular-grade water. thermal cycling was performed at • c for min for reverse transcription, followed by • c for min and then cycles of • c for s and • c for s. for testing the production of infectious virions, we titered the viruses as described in our previous studies [ ] [ ] [ ] . in summary, media from the viral culture were serially diluted from − to − in serum-free dmem containing . % bovine serum albumin. the dilutions were applied to a monolayer of vero-e cells in or -well plates. after one hour, cells were overlaid with virus growth medium containing % carboxymethyl cellulose and incubated for h. the cells were fixed and stained with crystal violet dye, and the plaques were calculated in each well and expressed as plaque-forming units per ml (pfu/ml). viral rna was extracted using the ntnu_mag_v protocol, a modified version of the bomb-protocol. libraries were prepared using a nugen trio rna-seq kit. sequencing was performed on the nextseq instrument (ns ; setup: pe × bp + single index ( bp)) using a nextseq mid sequencing kit and nextseq mid flow cell (ncs version: . . . ). reads were aligned using the bowtie software package version . . . to the reference viral genome wuhan-hu- / . sequence alignments were converted to binary alignments and sorted using samtools version . . the consensus fastq sequences were obtained with bcftools and vcfutils.pl (from samtools) and converted to fasta using seqtk (https://github.com/lh /seqtk). viral genomes in fasta formats were submitted to www.gisaid.org. the accession numbers of these genomes are: epi_isl_ (hcov- /norway/trondheim-e / ), epi_isl_ (hcov- /norway/trondheim-e / ), epi_isl_ (hcov- /norway/trondheim-s / ), epi_isl_ (hcov- /norway/trondheim-s / ), epi_isl_ (hcov- /norway/ trondheim-s / ), epi_isl_ (hcov- /norway/trondheim-s / ) and epi_isl_ (hcov- /norway/trondheim-s / ). transmission of the strains was visualized using https: //nextstrain.org/ncov/global?f_author=aleksandr% ianevski% et% al. the virus (multiplicity of infection, moi . ) was aliquoted in eppendorf tubes and incubated at − , − , , , and • c for h or at • c for min. alternatively, the virus was aliquoted in wells of a -well plate without a lid. the virus was exposed to uvc light (λ = nm, ≥ µw/cm ) for , , , , , and s using a germicidal lamp in a biosafety cabinet. the thermo and uvc stability of the viral rna was analyzed by rt-qpcr. subsequently, vero-e cells were infected with the virus for h, and cell viability was measured using a celltiter-glo assay (promega, madison, wi, usa). luminescence was read using a plate reader. approximately × vero-e cells were seeded per well in -well plates. the cells were grown for h in dmem supplemented with % fbs and pen-strep. serum samples were diluted -fold, and protein concentrations were quantified using nanodrop. serum samples were prepared in -fold dilutions at different concentrations, starting from mg/ml in the virus growth medium (vgm) containing . % bsa and pen-strep in dmem. virus hcov- /norway/trondheim-e / was added to the samples to achieve a moi of . and incubated for h at • c. . % dmso was added to the control wells. the vero-e cells were incubated for h with vgm. after the incubation period, the medium was removed, and a celltiter-glo assay was performed to measure viability. we have previously published a library of safe-in-man bsaas [ ] . supplementary materials table s lists these and other potential bsaas, their suppliers and catalogue numbers. the control wells. the cells were mock-or hcov- /norway/trondheim-e / -infected at a moi of . . after h of infection, the medium was removed from the cells, and a celltiter-glo assay was performed to measure viability. the half-maximal cytotoxic concentration (cc ) for each compound was calculated based on viability/death curves obtained on mock-infected cells after nonlinear regression analysis with a variable slope using graphpad prism software version . a (graphpad software, san diego, ca, usa). the half-maximal effective concentrations (ec ) were calculated based on the analysis of the viability of infected cells by fitting drug dose-response curves using the four-parameter ( pl) logistic function f (x): where f (x) is a response value at dose x, a min and a max are the upper and lower asymptotes (minimal and maximal drug effects), m is the dose that produces the half-maximal effect (ec or cc ) and λ is the steepness (slope) of the curve. a relative effectiveness of the drug was defined as the selectivity index (si = cc /ec ). the threshold of the si used to differentiate between active and inactive compounds was set to . area under the dose-response curve auc was quantified as: using the numerical integration implemented in the mess r package (bell laboratories, murray hill, nj, usa), where x max and x min are the maximal and minimal measured doses. serum sensitivity score (sss) was quantified as a normalized version of the standard auc (with the baseline noise subtracted and normalization of the maximal response at the highest concentration often corresponding to off-target toxicity) as where activity threshold t equals %. vero-e cells were treated with different concentrations of a combination of two bsaas. after h, cell viability was measured using a celltiter-glo assay. to test whether the drug combinations acted synergistically, the observed responses were compared with expected combination responses. the expected responses were calculated based on the zip reference model using synergyfinder web application, version [ ] . we measured the igg and igm in human serum using epitope diagnostics enzyme-linked immunosorbent assays (elisa) according to manufacturer specifications (epitope diagnostics, san diego, ca, usa). background-corrected optical density values were divided by the cutoff to generate signal-to-cutoff (s/co) ratios. samples with s/co values greater than . were considered positive. the pearson correlation coefficients were calculated by means of the stats r package, with the significance determined using a student's t-test. vero-e cells were treated with nelfinavir, amodiaquine or both drugs at indicated concentrations. cells were infected with the hcov- /norway/trondheim-e / strain at moi . or mock. after h, total rna was isolated using rneasy plus mini kit (qiagen, hilden, germany). libraries were prepared and sequenced on a nextseq (ns ) instrument (set up: pe × bp + single index bp) using a nextseq mid sequencing kit, nextseq mid flow cell, ncs version: . . . . reads were aligned using the bowtie software package version . . . to the ncbi reference sequence for sars-cov- (nc_ . ) and to the human grch genome. number of mapped and unmapped reads that aligned to each gene were obtained with the featurecounts function from rsubread r-package version . . the gff table for the reference sequence was downloaded from https://ftp.ncbi.nlm.nih.gov/genomes/all/gcf/ / / /gcf_ . _asm v /gcf_ . _asm v _genomic.gff.gz and flattened to gtf format and given as an additional argument to the rsubread function. the heatmaps were generated using the pheatmap package (https://cran.r-project.org/web/packages/pheatmap/index.html) based on log -transformed profiling data. we reviewed the current landscape of the available diagnostic tools, as well as the emerging treatment and prophylactic options for the sars-cov- pandemic and have summarized the information in a database that can be freely accessed at https://sars-coronavirus- .info. the information in the database was obtained from pubmed, clinicaltrials.gov, drugbank, drugcentral, the chinese clinical trials register (chictr) and eu clinical trials register databases [ ] [ ] [ ] , as well as other public sources. the website was developed with php v technology using d .js v (https://d js.org/) for visualization. the covid- statistics are automatically exported from the covid- data repository by the center for systems science and engineering (csse) at johns hopkins university (https://github.com/cssegisanddata/covid- ). we isolated seven sars-cov- strains from nps samples of covid- patients using vero-e cells. the rt-qpcr cycle threshold (ct) values were - before and - after propagation of the viruses in vero-e cells (figure a ,b). we sequenced seven strains and found that the sequences differed from the reference hcov- /wuhan/wiv / strain by a few missense mutations ( figure c) . phylogenetic analysis showed a close relationship between the strains (figure d ). in cross-referencing our sequence data with the pathogen-tracking resource nextstrain.org, we determined that the sars-cov- strains isolated in trondheim originated from china, denmark, the usa and canada (figure e) . in addition, we tested several cell lines and found that vero-e was the most susceptible for virus-mediated death and virus amplification (figure s a-c). to establish the rationale for safe work, we incubated the virus at different temperatures for h or exposed to uvc radiation for different time periods. the resulting virus preparations were analyzed by rt-qpcr and used to infect vero-e cells. virus incubation at • c for h or uvc exposure for sec destabilized the virus and rescued infected cells from virus-mediated death ( figure s ). we evaluated the neutralization capacity of seven serum samples obtained from patients recovered from covid- . we also used sera from patients recovered from endemic coronavirus infections and from healthy blood donors as controls. five samples from patients recovered from covid- had serum sensitivity scores (sss) > and rescued > % cells from virus-mediated death at mg/ml (figure a,b) . notably, that serum from a recovered patient with sss = . neutralized all seven sars-cov- strains ( figure s ). our neutralization test of samples (table s ) showed a moderate positive correlation with igg (r = . , p < . ) and igm (r = . , p = . ) s/co values obtained using commercial elisa kits that recognize the sars-cov- n protein (figure c,d) . however, the correlation between the igg and igm elisa results was only r = . , p = . . furthermore, we found a moderate negative correlation between ssss and time intervals between the sars-cov- diagnosis and serum collection for samples (- . , p < . ; figure d ). altogether, these results suggest that patients diagnosed with covid- produce different immune responses to the sars-cov- infection and that the neutralization capacity of convalescent sera declines with time. we evaluated the neutralization capacity of seven serum samples obtained from patients recovered from covid- . we also used sera from patients recovered from endemic coronavirus infections and from healthy blood donors as controls. five samples from patients recovered from covid- had serum sensitivity scores (sss) > and rescued > % cells from virus-mediated death at mg/ml (figure a,b) . notably, that serum from a recovered patient with sss = . neutralized all seven sars-cov- strains ( figure s ). our neutralization test of samples (table s ) showed a moderate positive correlation with igg (r = . , p < . ) and igm (r = . , p = . ) s/co values obtained using commercial elisa kits that recognize the sars-cov- n protein (figure c,d) . however, the correlation between the igg and igm elisa results was only r = . , p = . . furthermore, we found a moderate negative correlation between ssss and time intervals between the sars-cov- diagnosis and serum collection for samples (− . , p < . ; figure d ). altogether, these results suggest that patients diagnosed with covid- produce different immune responses to the sars-cov- infection and that the neutralization capacity of convalescent sera declines with time. through the literature review, we made a database to summarize safe-in-man bsaas (https://drugvirus.info/). recently, we have expanded on the spectrum of activities for some of these agents [ ] [ ] [ ] , ] . some of these agents could be repositioned for the treatment of a sars-cov- infection. we tested agents against sars-cov- in vero-e cells. remdesivir was included as a positive control [ ] and nicotine as a negative control. seven different concentrations of the compounds were added to virus-infected cells. cell viability was measured after h to determine compound efficiency. after the initial screening, we identified apilimod, emetine, amodiaquine, obatoclax, homoharringtonine, salinomycin, arbidol, posaconazole and nelfinavir as compounds that rescued virus-infected cells from death (auc from to ; table s ). the compounds we identified possessed a structure-activity relationship (figure a) . auc for remdesivir was . interestingly, µm of nicotine rescued cells from virus-mediated death but altered the cell morphology (auc = ; figure s ). we repeated the experiment with hit compounds, monitoring their toxicity and efficacy. we confirmed the antiviral activity of emetine, amodiaquine, obatoclax, homoharringtonine, salinomycin and nelfinavir (figure b ,c). importantly, amodiaquine had a superior si over its analogs chloroquine, hydroxychloroquine, quinacrine and mefloquine ( figure s ). thus, we identified and validated anti-sars-cov- activities for six bsaas in vero-e cells. to test for potential synergism among the validated hits, we treated cells with varying concentrations of a two-drug combination and monitored the cell viability (figure a) . the observed drug combination responses were compared with the expected combination responses calculated by means of the zero-interaction potency (zip) model [ , ] . we quantified synergy scores, which represent the average excess response due to drug interactions (i.e., % of cell survival beyond the expected additivity between single drugs has a synergy score of ). we found that combinations of nelfinavir with salinomycin, amodiaquine, homoharringtonine and obatoclax, as well as the combination of amodiaquine and salinomycin, were synergistic (most synergistic area scores > ; figure b ). moreover, the nelfinavir-amodiaquine treatment was effective against all seven sars-cov- strains (figure c ). thus, we identified synergistic drug combinations against sars-cov- infections. we next profiled transcriptional responses to nelfinavir, amodiaquine or both drugs in virus-or mock-infected vero-e cells at h. we showed that the addition of nontoxic but effective concentrations of drugs slightly affected the transcription of immune-related genes in virus-infected cells ( figure s a ). these genes (cxcl , cxcl , cxcl , cxcl , cxcl , cxcl , oasl, ifnl , mx and herc ) are needed for alarming neighboring cells about the ongoing infection and for the protection of the organism from repeated infections. amodiaquine and its combination with nelfinavir lowered the transcription of viral genomic and sub-genomic rnas ( figure s b) . (a) structure-antiviral activity relation of broad-spectrum antivirals (bsaas). the compounds were clustered based on their structural similarity calculated by ecpf fingerprints and visualized using the d javascript library. the anti-sars-cov- activity of the compounds was quantified using the auc and shown as bubbles. bubble size corresponds to compounds aucs. (b) vero-e cells were treated with increasing concentrations of a compound and infected with the hcov- /norway/trondheim-e / strain (moi, . ) or mock. after h, the viability of the cells was determined using the celltiter-glo assay. mean ± sd; n = . (c) table showing half-maximal cytotoxic concentration (cc ), the half-maximal effective concentration (ec ) and selectivity indexes (si = cc /ec ) for selected anti-sars-cov- compounds calculated from ctg and plaque assays. mean ± sd; n = . figure . anti-sars-cov- activity of safe-in man broad-spectrum antivirals in vero-e cells. (a) structure-antiviral activity relation of broad-spectrum antivirals (bsaas). the compounds were clustered based on their structural similarity calculated by ecpf fingerprints and visualized using the d javascript library. the anti-sars-cov- activity of the compounds was quantified using the auc and shown as bubbles. bubble size corresponds to compounds aucs. (b) vero-e cells were treated with increasing concentrations of a compound and infected with the hcov- /norway/trondheim-e / strain (moi, . ) or mock. after h, the viability of the cells was determined using the celltiter-glo assay. mean ± sd; n = . (c) table showing half-maximal cytotoxic concentration (cc ), the half-maximal effective concentration (ec ) and selectivity indexes (si = cc /ec ) for selected anti-sars-cov- compounds calculated from ctg and plaque assays. mean ± sd; n = . to rapidly respond to the covid- outbreak, we developed a freely accessible website summarizing novel anti-sars-cov- developments and currently approved diagnostic options around the globe. it also tracks the development of therapeutic/antiviral drugs and vaccines. the "treatment" section of the website summarizes in-progress and completed clinical trials that test the efficacy of therapeutic agents to treat covid- or complications that arise from covid- . these trials include over unique therapeutic agents in varying combinations and applications. importantly, we list clinical trials that are already completed or are projected to be completed by the end of june . of note, among these are trials of remdesivir, favipiravir, lopinavir/ritonavir, hydroxychloroquine, to rapidly respond to the covid- outbreak, we developed a freely accessible website summarizing novel anti-sars-cov- developments and currently approved diagnostic options around the globe. it also tracks the development of therapeutic/antiviral drugs and vaccines. the "treatment" section of the website summarizes in-progress and completed clinical trials that test the efficacy of therapeutic agents to treat covid- or complications that arise from covid- . these trials include over unique therapeutic agents in varying combinations and applications. importantly, we list clinical trials that are already completed or are projected to be completed by the end of june . of note, among these are trials of remdesivir, favipiravir, lopinavir/ritonavir, hydroxychloroquine, dipyridamole and interferons alpha and beta, which are all phase or clinical trials scheduled to be currently completed. the "prevention" section summarizes current vaccine trials taking place around the globe. although vaccine development lags considerably behind drug development, several repurposed vaccine options have also emerged. this includes trials of the cross-reactivity of the mmr (measles, mumps and rubella) vaccine, as well as several trials of the bacillus calmette-guérin (bcg) vaccine among high-risk populations, such as healthcare workers. finally, the "testing" section of the website provides a summary of currently available laboratory-based and point-of-care diagnostic tests that are approved for clinical diagnosis in at least one country. the website also includes predictions of experimental and approved drugs effective against sars-cov- , as well as provides a summary of information about the coronavirus pandemic. the website allows interactive exploration of the data with built-in feedback and is available in several languages. the website is updated as soon as novel anti-sars-cov- options emerge, or the statuses of existing ones are updated. here, we reported the isolation of seven sars-cov- strains from samples of patients suffering from covid- . full-genome sequencing revealed that the strains were highly similar ( . %) to one another and to the strains circulating in china, denmark, the usa and canada. all seven strains contain d g in the s protein. strains with this mutation began spreading in europe in early february and became dominant in other regions (https://doi.org/ . / . . . ). we screened cell lines for their susceptibility for the sars-cov- infection and virus replication. vero-e cells appeared to be the most susceptible cell line for virus-mediated cell death and virus propagation. this cell line has been widely used in toxicology, virology and pharmacology research, as well as in the production of vaccines and diagnostic reagents. the cell line is interferon-deficient; i.e., unlike normal mammalian cells, it does not secrete interferon alpha or beta when infected by viruses [ ] . moreover, this cell line was used routinely in anti-sars-cov- research [ ] [ ] [ ] [ ] . we also showed that > sec of uvc radiation or h incubation at • c neutralized sars-cov- , establishing a rationale and methodology for safe work in the laboratory. these results are consistent with previous studies showing that physical factors destabilize sars-cov- and other viruses [ ] [ ] [ ] [ ] . neutralization tests are crucial tools for the assessment of previous sars-cov- exposure and potential immunity [ , ] . we have developed a test to assess the neutralization capacity of serum samples from patients recovered from sars-cov- infections, patients with endemic coronavirus infections and healthy blood donors. our results suggest that covid- patients respond differently to the sars-cov- infection. moreover, the neutralization capacity of convalescence sera declined with time. thus, the neutralization test allowed us to identify the most potent sera from patients recovered from covid- for the treatment of sars-cov- -infected patients. moreover, results from our neutralization test positively correlated with those from commercial elisa assays. however, the correlation between the igg and igm elisa results was only moderate. the difference could be associated with the time of the sample collection, production of the immunoglobulins or sensitivity that can be attributed to the technique and the antigen in use (i.e., igm is the first immunoglobulin to be produced in response to an antigen and can be detected during early onset of disease, whereas igg is maintained in the body after initial exposure for the long-term response and can be detected after the infection has passed). there were certain concerns regarding the antibody-dependent cell-mediated toxicity of convalescent sera [ ] . however, the recent safety study on hospitalized patients transfused under the u.s. food and drug administration's national expand access program for covid- revealed no toxicity (https://doi.org/ . / . . . ) . drug repurposing, also called drug repositioning, is a strategy for generating an additional value from an existing drug by targeting diseases other than that for which it was originally intended [ , ] . this has significant advantages over new drug discoveries, since chemical synthesis steps, manufacturing processes, reliable safety and pharmacokinetic properties have already been studied in preclinical (animal model) and early clinical developmental phases (phase , i and iia). therefore, drug repositioning for covid- provides unique translational opportunities, including a substantially higher probability of success to the market as compared with developing new virus-specific drugs and vaccines, as well as significantly reduced costs and timelines to clinical availability [ , , ] . we tested safe-in-man bsaas against sars-cov- in cell cultures. we identified salinomycin, obatoclax, amodiaquine, nelfinavir, emetine and homoharringtonine as having anti-sars-cov- activity, which we put forward as potential anti-sars-cov- drug candidates. nelfinavir (viracept) is an orally bioavailable inhibitor of human immunodeficiency virus hiv- ( mg per os (po) q hr). it targets hiv protease for the treatment of hiv infections [ ] . molecular docking studies predict that nelfinavir binds to the sars-cov- protease [ ] . nelfinavir could also inhibit cell fusion caused by the sars-cov- s glycoprotein (https://doi.org/ . / . . . ) [ ] . it also inhibits chikungunya virus (chikv), dengue virus (denv), hepatitis c virus (hcv), herpes simplex virus (hsv- ) and sars-cov infections (https://doi.org/ . / c ra h) [ ] [ ] [ ] . amodiaquine is a medication used to treat malaria. the recommended dose for a course of amodiaquine is mg amodiaquine base/kg body weight over three days, i.e., mg/kg/day [ ] . [ ] [ ] [ ] [ ] [ ] [ ] . importantly, amodiaquine showed more potent antiviral activity than its analogs chloroquine and hydroxychloroquine. obatoclax was originally developed as an anticancer agent. several phase ii clinical trials have investigated the use of obatoclax in the treatment of leukemia, lymphoma, myelofibrosis and mastocytosis. a continuous h infusion of obatoclax - mg/day for three days in two-week cycles or h infusions in a -day cycle have previously been evaluated in cancer patients [ ] . in addition, obatoclax showed antiviral activity against fluav, zikv, wnv, yfv, sinv, junín virus (junv), lasv, herpes simplex virus (hsv- ), echovirus (ev ), human metapneumovirus (hmpv), rvfv and lymphocytic choriomeningitis virus (lcmv) in vitro [ , , , , ] . it was shown that obatoclax inhibited the viral endocytic uptake by targeting the cellular mcl- protein [ ] . emetine is an antiprotozoal drug. it is administered by intramuscular or deep subcutaneous injection in a dose of mg/kg/day (maximum mg/day) for days [ ] . emetine is also used to induce vomiting. in addition, it possesses antiviral effects against zikv, ebov, rabv, cytomegalovirus (cmv), hcov-oc , hsv- , ev , hmpv, rvfv, fluav, hiv- and sars-cov- [ , , , [ ] [ ] [ ] [ ] [ ] (https://doi.org/ . / . . . ). emetine was proposed to inhibit viral polymerases, though it could have some other targets [ ] . homoharringtonine is an anticancer drug that is indicated for the treatment of chronic myeloid leukemia ( mg/m iv daily × ). it also possesses antiviral activities against hepatitis b virus (hbv), mers-cov, hsv- , ev , vzv and sars-cov- in vitro [ , , [ ] [ ] [ ] [ ] . homoharringtonine binds to the s ribosome and inhibits viral protein synthesis by interfering with the chain elongation [ ] . salinomycin is an orally bioavailable antibiotic that is used against gram-positive bacteria in animal husbandry ( . mg/kg body weight (bw) po). it also inhibits fluav, respiratory syncytial virus (rsv) and cmv infections [ , ] . salinomycin was proposed to disrupt the endosomal acidification and to block entry of the viruses into cells [ , ] . our results have uncovered several existing bsaas that could be repositioned to sars-cov- infections. since pharmacokinetic/pharmacodynamic and toxicology studies have already been performed on these compounds, in vivo efficacy studies could be initiated immediately, saving time and resources. combination therapies became a standard for the treatment of hiv and hcv infections. the reasons for using combinations rather than single antiviral are better efficacy, decreased toxicity and the prevention of resistance emergence. here, we found that combinations of nelfinavir with salinomycin, amodiaquine, obatoclax, emetine or homoharringtonine were synergistic against sars-cov- in vero-e cells. interestingly, synergistic interactions occurred between compounds belonging to different sar clusters (i.e., nelfinavir belongs to a separate cluster than amodiaquine or obatoclax-emetine-homoharringtonine). in particular, the synergy was achieved when a virus-directed drug was combined with host-directed ones. this observation agrees with other studies on such combinations and virus-host interactions [ , , , ] (https://doi.org/ . / . . . ). according to the available pharmacological data for these drugs, the most potent combination could be a combination of orally available nelfinavir and amodiaquine. this was also the combination that exhibited the highest synergy of all the drug combinations we tested, with the synergy score at the most synergistic area being . (i.e., . % of cell survival beyond the expected additivity between the single drugs). there are no guidelines of what is considered a good synergy, but it is very common to consider synergy > as true (significant) synergy. thus, the amodiaquine and nelfinavir combination could result in better efficacy and decreased toxicity for the treatment of sars-cov- and perhaps other viral infections. our future goal is to complete preclinical studies and translate our findings into trials in patients. the most effective and tolerable bsaas or their combinations will have a global impact, improving the protection of the general population from emerging and re-emerging viral infections or coinfections and allowing the rapid management of drug-resistant strains. our bigger ambition is to assemble a toolbox of bsaas for the treatment of emerging and re-emerging viral infections. this toolbox can be offered to the who as a means for the fast identification of safe and effective antiviral options. we have summarized the information about the status of currently available and emerging anti-sars-cov- options on the freely accessible website (https://sars-coronavirus- .info). the website is updated regularly and incorporates novel anti-sars-cov- options as they emerge or changes the statuses of existing ones as updates occur. in conclusion, sera from recovered patients, bsaas and combinations of bsaas, as well as other available and emerging treatments, could have pivotal roles in the battle against covid- and other emerging and re-emerging viral diseases. further development of these options could save time and resources that are required for the development of alternative virus-specific drugs and vaccines. this could have a global impact by decreasing morbidity and mortality, maximizing the number of healthy life years, improving the quality of life of infected patients and decreasing the costs of patient care curtailing to the impact of the current sars-cov- pandemic, as well as future viral outbreaks. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s : table s : compounds, their suppliers, catalogue numbers and aucs. table s . results of neutralization and elisa assays. figure s . propagation of hcov- /norway/trondheim-e / in cell cultures. figure s . effects of temperature and uv radiation on the infectivity of the hcov- /norway/trondheim-e / strain. figure s . the effects of the serum from patients recovered from the sars-cov- infection on the viability of vero-e cells infected with sars-cov- strains. figure s . the effects of nicotine on the viability and morphology of mock-and sars-cov- -infected vero-e cells. figure s : the comparison of anti-sars-cov- activities of amodiaquine and its analogs. figure s . transcriptomic analysis of mock-and sars-cov- -infected vero-e cells treated with nelfinavir, amodiaquine or both drugs. author contributions: all authors contributed to the methodology, software, validation, formal analysis, investigation, resources, data curation, writing and review and editing of the manuscript. d.k. conceptualized, supervised and administrated the study and acquired funding. all authors have read and agreed to the published version of the manuscript. funding: this research was funded the european regional development fund, the mobilitas pluss project mobtt (to d.k.). available online: www.who.int/medicines/ebola-treatment/who-list-of-top-emerging-diseases/en emerging virus diseases: can we ever expect the unexpected? emerg. microbes infect. , , e infectious disease. combating emerging viral threats srebp-dependent lipidomic reprogramming as a broad-spectrum antiviral target experimental drugs poised for use in ebola outbreak an off-target effect of bx blocks herpes simplex virus type infection of the eye repurposing of kinase inhibitors as broad-spectrum antiviral drugs the future of antivirals: broad-spectrum inhibitors approaches for identification of hiv- entry 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coronavirus (sars-cov- ) in cell culture remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov- replication in vitro lianhuaqingwen exerts anti-viral and anti-inflammatory activity against novel coronavirus (sars-cov- ) repurposing of clinically approved drugs for treatment of coronavirus disease in a -novel coronavirus-related coronavirus model temperature decreases spread parameters of the new covid- case dynamics effectiveness of ultraviolet-c light and a high-level disinfection cabinet for decontamination of n respirators association between climate variables and global transmission of sars-cov- low temperature and low uv indexes correlated with peaks of influenza virus activity in northern europe during antibody-dependent cell-mediated cytotoxicity against influenza virus-infected cells editorial: drug repositioning: current advances and future perspectives drug repurposing: progress, challenges and recommendations drug repurposing approaches for the treatment of influenza viral infection: reviving old drugs to fight against a long-lived enemy drug repurposing screens and synergistic drug-combinations for infectious diseases circulating metabolites of the human immunodeficiency virus protease inhibitor nelfinavir in humans: structural identification, levels in plasma, and antiviral activities binding site analysis of potential protease inhibitors of covid- using autodock the anti-hiv drug nelfinavir mesylate (viracept) is a potent inhibitor of cell fusion caused by the sarscov- spike (s) glycoprotein warranting further evaluation as an antiviral against covid- infections nelfinavir inhibits maturation and export of herpes simplex virus inhibition of intracellular hepatitis c virus replication by nelfinavir and synergistic effect with interferon-alpha hiv protease inhibitor nelfinavir inhibits replication of sars-associated coronavirus amodiaquine dosage and tolerability for intermittent preventive treatment to prevent malaria in children identification of broad-spectrum antiviral compounds by targeting viral entry arbidol and other low-molecular-weight drugs that inhibit lassa and ebola viruses novel amodiaquine derivatives potently inhibit ebola virus infection high-content screening in hpsc-neural progenitors identifies drug candidates that inhibit zika virus infection in fetal-like organoids and adult brain repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection amodiaquine, an antimalarial drug, inhibits dengue virus type replication and infectivity a multicenter phase i/ii study of obatoclax mesylate administered as a -or -hour infusion in older patients with previously untreated acute myeloid leukemia the reframe library as a comprehensive drug repurposing library to identify mammarenavirus inhibitors obatoclax inhibits alphavirus membrane fusion by neutralizing the acidic environment of endocytic compartments a phase i study of emetine hydrochloride (nsc ) in solid tumors high-throughput screening and identification of potent broad-spectrum inhibitors of coronaviruses retrograde axonal transport of rabies virus is unaffected by interferon treatment but blocked by emetine locally in axons emetine inhibits zika and ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry efficacy and mechanism of action of low dose emetine against human cytomegalovirus natural plant alkaloid (emetine) inhibits hiv- replication by interfering with reverse transcriptase activity emetine inhibits replication of rna and dna viruses without generating drug-resistant virus variants anti-varicella-zoster virus activity of cephalotaxine esters in vitro the natural compound homoharringtonine presents broad antiviral activity in vitro and in vivo a screen of the nih clinical collection small molecule library identifies potential anti-coronavirus drugs effect of cantharidin, cephalotaxine and homoharringtonine on "in vitro" models of hepatitis b virus (hbv) and bovine viral diarrhoea virus (bvdv) replication targeting intracellular ion homeostasis for the control of respiratory syncytial virus wnt modulating agents inhibit human cytomegalovirus replication salinomycin inhibits influenza virus infection by disrupting endosomal acidification and viral matrix protein function identification of antiviral drug candidates against sars-cov- from fda-approved drugs jnj inhibits influenza a virus replication without altering cellular antiviral responses in vitro testing of combined hydroxychloroquine and azithromycin on sars-cov- shows synergistic effect acknowledgments: this study is devoted to wenliang li, a chinese doctor who tried to warn about coronavirus, as well as to many other doctors and covid- patients. we thank koit aasumets, sergio miguel castañeda zegarra, qindong zhang, simona komarova, nikki upfold, miriam khider, hege hval and kasia kolasa for translation of the sars-coronavirus- .info website to different languages. we also thank maxim bespalov, sergei shiryaev, pavel uvarov, evgeny kulesskiy and many other people for sharing their ideas on drug candidates. in addition, we thank wei wang, marit bugge and nadra j. nilsen for the cell lines. the authors declare no conflicts of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -nyyunoha authors: orlinger, klaus k.; holzer, georg w.; schwaiger, julia; mayrhofer, josef; schmid, karl; kistner, otfried; noel barrett, p.; falkner, falko g. title: an inactivated west nile virus vaccine derived from a chemically synthesized cdna system date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: nyyunoha a cdna comprising the complete genome of west nile virus (wnv) was generated by chemical synthesis using published sequence data, independent of any preformed viral components. the synthetic wnv, produced by transfection of in vitro transcribed rna into cell culture, exhibited undistinguishable biological properties compared to the corresponding animal-derived wild-type virus. no differences were found concerning viral growth in mammalian and insect cell lines and concerning expression of viral proteins in cells. there were also no significant differences in virulence in mice following intranasal challenge. after immunizations of mice with experimental vaccines derived from the synthetic and wild-type viruses, protection from lethal challenge was achieved with similar amounts of antigen. both vaccine preparations also induced comparable levels of neutralizing antibodies in mice. in addition, the synthetic approach turned out to be very accurate, since the rescued wnv genome contained no undesired mutations. thus, the first flavivirus based on chemical gene synthesis was indistinguishable from the parent virus. this demonstrates that virus isolates from animal sources are dispensable to derive seed viruses for vaccine production or research. west nile virus (wnv) is a mosquito-borne, neurotropic member of the genus flavivirus, family flaviviridae, and has been identified in africa, europe, the middle east, south and central asia, oceania (subtype kunjin), and most recently north america (reviewed in [ ] ). in the u.s. wnv activity in human, bird, companion animals or mosquito has been reported since to the centers for disease control (cdc) from almost all states. besides wnv, the genus flavivirus comprises a number of medically important pathogens including japanese encephalitis virus (jev), yellow fever virus (yfv), tick borne encephalitis virus (tbev) and the four serotypes of dengue virus (denv) [ ] . the flavivirus genome is a positive-polarity, single-stranded rna molecule of about , nucleotides (nt), which functions as mrna for translation of the viral proteins. genomic rna is infectious when introduced into susceptible cells by transfection [ ] . for replication and pathogenesis studies, reverse genetic systems have been established for several members of the genus [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these systems comprise one or two plasmids encoding cdna of viral genomic sequence under control of bacteriophage promoters allowing transcription of full-length infectious rna in vitro. for yfv [ ] , den- [ ] , den- [ , , ] , den- [ ] , tbev [ , ] , kun [ ] , mve [ ] and wnv lineage i [ ] and ii [ ] , cdna comprising the full genome was stably cloned into bacterial expression plasmids, whereas in other reports [ , , , , ] cdna was split in two fragments, each integrated in individual plasmids, from which cdna can be fused together before rna transcription. an alternative approach was applied to construct a jev infectious clone, in which the viral coding sequence was put under the control of a eukaryotic promoter and split by introns to circumvent instability during propagation in bacteria [ ] . conventional generation of such cdna clones requires the production of an initial virus stock, viral rna isolation, reverse transcription, pcr amplification of subfragments and engineering into the final transcription units. these approaches are sometimes hampered by low fidelity of reverse transcriptase or sequence variations in the starting isolate, which may lead to undesired alterations of the genomic sequence. as a consequence, in most reports in which the viral cdna clones or generated viruses were analyzed by sequence analysis, nucleotide variations were detected compared to the published sequence of the parent virus [ , , , , , , ] . in , a landmark publication proved the feasibility of de novo synthesis of a poliovirus by biochemical synthesis precluding any preformed components. the viral cdna encoding the . kb genome was assembled from overlapping oligonucleotides and yielded infectious virus after transcription of genomic rna and inoculation into cell lysates [ ] . taking advantage of the rapid progression of gene synthesis technology (for review [ ] ), we intended to adopt such a synthetic approach to produce a flavivirus cdna system for the generation of a synthetic wnv seed virus for use in vaccine development. in this study we report the generation of a fully functional wnv virus from a completely synthetic source. the whole , -nucleotide wnv genomic sequence was generated by gene synthesis without using natural viral templates. the production and characterization of the resulting west nile virus, which fully matched the sequence of the in silico designed viral genome, confirms the feasibility and accuracy of the synthetic flavivirus reverse genetic system. wnv wild-type virus strain ny -flamingo - was obtained from centers for disease control (cdc, atlanta) corresponding to genbank accession #af . this sequence information was also used as template for in silico design for de novo synthesis of the genomic cdnas. the cell lines vero (atcc ccl- ), bhk (atcc ccl- ) and c - (eceacc .p. # d ) were obtained from the american type culture collection or european collection of cell cultures and grown in dubecco's modified eagle's medium (dmem) or tc-vero media (baxter). tc-vero is an animal protein-free medium based on dmem/ham's f medium. six dna fragments corresponding to wnv strain ny -flamingo - (genbank accession #af ) were generated by chemical synthesis (geneart, regensburg, germany). plasmid p tl-ab carried dna corresponding to wnv genomic sequence nt - , plasmid p tl-cd to nt - , plasmid p tl-ab to nt - , plasmid p tl-cd to nt - , plasmid p tl-ef to nt - , and plasmid p tl-gh to nt , - , . three silent mutations at positions , , and were introduced to knock out an ecori site and to create an styi site as described previously [ ] . for assembly of plasmid pwnvsyn- tl (containing the third of the wnv coding sequence) an asci and bsai cut fragment of p tl-ab was ligated to a bbsi and paci cut fragment of p tl-cd (fig. b) . the ligation product was amplified using high fidelity pcr (kod, invitrogen). following digestion with bamhi and xbai, the fragment was cloned into the low copy plasmid vector pbr -pl (derived from plasmid pbr engineered to contain a matching polylinker between the unique restriction sites ecor and eagi resulting in the partial deletion of the tet r gene) yielding pwnvsyn- tl. this plasmid contains the two-thirds of the wnv coding sequence and was generated in three steps: (i) an asci and bsai cut fragment of p tl-ab was ligated to a bsai and paci cut fragment of p tl-cd. this fragment was amplified by high fidelity pcr and integrated into a commercially available ppcrscript vector (clontech). (ii) a btgzi and asci cut fragment of p tl-ef was ligated to a paci and btgzi cut fragment of p tl-gh. the resulting tl-efgh ligation product was amplified by pcr using and flanking primers, digested with spei and xbai and integrated in pbr -pl, leading to plasmid pbr - tl efgh (fig. b) . (iii) the final pwnvsyn- tl was generated by introduction of the tl-abcd fragment (derived from ppcrscript tl-abcd) into pbr - tl-efgh, taking advantage of the unique restriction enzymes spei and bamhi (fig. c) . all plasmids were amplified in bacterial strain hb (promega) and purified with commercially available systems (omega and qiagen). electroporation of bacterial cells was carried out using a genepulser apparatus (bio-rad) with settings of . kv, f and . sequence analysis was carried out using a xl genetic analyzer (abi) using bigdye terminator v . cycle sequencing kit (abi). pwnvsyn- tl was linearized with xbai followed by mung bean nuclease digestion to remove single stranded nucleotide overhangs in order to generate the correct end of the wnv coding sequence. the plasmids pwnvsyn- tl and pwnvsyn- tl were then digested with sphi and the full-length sequence was generated by ligation (t ligase; new england biolabs) via the sphi sequence overhangs of the and parts. the ligated dna fragments were extracted with phenol-chloroform twice, precipitated with ethanol and resuspended in nuclease free water. rna was transcribed at • c for h from ligated template dna by t polymerase transcription, using t megascript kit (ambion). the integrity of rna transcripts was analyzed in % agarose gels containing % formaldehyde. for rna transfection, subconfluent vaccine-certified vero cells were collected with trypsin, washed twice in serum free tc vero medium (baxter) and twice in ice-cold pbs buffer. aliquots of approximately × cells were resuspended in l of ice-cold pbs, mixed with transcribed rna and transferred to . gene pulser cuvettes. cells were electroporated with three successive pulses using a genepulser apparatus (bio-rad) with settings of . kv, f and . to visualize intracellular expression of wnv proteins, cells were infected or transfected. two days later, cells were fixed with acetone-methanol ( : ). cover slips with fixed cells were dried, rehydrated with phosphate-buffered saline and treated with a polyclonal mouse anti-wnv serum ( : dilution) obtained after immunization of mice with a formalin-inactivated whole virus vaccine preparation. bound antibodies were visualized with fluorescein isocyanate-conjugated anti-mouse immunoglobulin ( : dilution; jackson research laboratory). vero or c / cells grown in cm tissue culture flasks were infected with either wnvsyn or wnvwt stock at an moi of . . the inoculum was removed after h, and ml of fresh medium was added. at various time points ( , , , , , and h) . ml of medium was removed. the infectious virus titer of wnv containing samples was determined by a tcid assay. in brief, serial -fold dilutions of virus containing supernatant were inoculated in -well microtiter plates seeded with vero cells. after incubation for days at • c and % co , the plates were screened under a light microscope for the presence of cpe in individual wells. from the number of cpe positive wells per dilution step, the tcid was calculated according to the poisson formula by means of an in house calculation software program. viral rna was extracted from supernatant containing viral material corresponding to × tcid by trizol extraction. rna was precipitated with ethanol and the rna pellet was resuspended in l of nuclease-free water. one l of rna was used for cdna transcription using superscript iii cdna synthesis kit (invitrogen) and primers binding in the end of the ns coding region, the ns b coding region and the noncoding region. for the generation of inactivated whole virus vaccines, the wnvsyn and wnvwt stocks were amplified on bhk cells to serve as prime/boost antigen in animal studies. the wnvsyn preparation (designated cag ) as well as wnvwt preparation (designated cag ) was prepared in the same manner. ten roller bottles of bhk cells were infected with a moi of . . for better virus yields ph was adjusted to . after h of virus adsorption. after days of growth the supernatant was harvested and cleared through a low spin centrifugation step at rpm. the cleared supernatant was treated with formalin (final concentration . %) for h. next, ml of the inactivated virus was loaded on ml of a % sucrose cushion per centrifugation tube (beckman, sw tubes). after h centrifugation with , × g the supernatant was discarded and resulting pellets were pooled in tris buffered saline (tbs). an aliquot of the resulting vaccine preparations was subjected to a safety assay to exclude any possible remaining infectivity. in order to normalize the administered amounts of antigen given in immunogenicity studies, the antigen content was determined in a wnv-specific antigen elisa. the lethal dose (ld ) was determined in female -weekold balb/c mice. groups of six mice were infected intranasally with × , × , × and × tcid of wnvsyn or wnvwt, respectively. survival of mice was recorded for a period of days after infection. the -fold virus dilutions were titrated shortly after challenge and were used to calculate the ld values using the computer program graph pad prism . protection was determined after immunization of female -week-old balb/c mice by subcutaneous injections of formalininactivated wnvsyn or wnvwt vaccines in a volume of l in tbs containing . % al(oh) . mice were challenged intranasally with l of pbs ( . % human serum albumin) containing × tcid wnvwt virus. survival was monitored over a period of days after challenge. for neutralizing antibody determination, serum samples were serially diluted with cell culture medium in twofold steps. the serum dilutions were mixed at a ratio of : with a virus stock suspension adjusted to × tcid , incubated for ± min at room temperature and transferred (eight replicates per dilution) to a -well microtiter plate seeded with vero cells. the plates were inspected under a light microscope for the presence of cpe after incubation for days at • c and % co . the neutralizing titer was calculated by counting cpe negative wells and by usage of the formula nt-titer = (v/ ) × e((nneg/ ) + . ) whereas nneg is the amount of negative wells and v represents the dilution of the sera in the neutralization mix. for each assay a defined serum positive control was measured and the titer of the viral material was titrated. for detecting infectious viral material in formalin-inactivated wnv antigen preparations, vero and c / cells were seeded in five cm tissue culture flasks and inoculated with individual preparations corresponding to ml of the infectious yield from which the preparations were derived. after a day incubation period at • c and % co , supernatant of each flask was titrated by tcid and ml supernatant of each flask was carried onto fresh vero and c / cells. after a -day observation period supernatant of each flask was titrated by tcid . the respective antigen preparations were classified as safe, when no cpe was detectable in individual flasks and no viral material was detected in both tcid assays. the amount of wnv antigen in respective samples was determined by means of an elisa double sandwich system. briefly, -well microtiter plates were coated by overnight incubation at - • c with an anti-wnv igg polyclonal serum raised in guinea pigs. after subsequent washing steps, serial twofold dilutions of a standard west nile virus production material (wnv peak pool) with defined antigen amount, a control sample and individual samples were applied to the microtiter plate which was then incubated for h at • c. after subsequent washing steps a mouse anti-wnv polyclonal serum was applied to the wells and incubated for h at • c. after washing, the wells were incubated with horseradish peroxidase-conjugated donkey anti-mouse igg (jackson immuno research laboratories) for h at • c. after subsequent washing steps, substrate (o-phenylenediamine/h o ) was added, and the enzyme reaction was stopped after min at • c by the addition of . m h so . the absorbance at nm was measured with an elisa plate reader (bio-tek, winooski, vt, usa) and the antigen content was calculated (kc software; bio-tek) by means of the standard curve derived from the dilution steps of the wnv peak pool standard material. all animal experiments were reviewed by the institutional animal care and use committee (iacuc) and approved by the austrian regulatory authorities and were conducted in accordance with austrian laws on animal experimentation and guidelines set out by the association for assessment and accreditation of laboratory animal care (aaalac). animals were housed in facilities accredited by the aaalac. all experiments with infectious virus were carried out under biosafety level conditions. experiments were approved by the baxter internal biosafety committee and by the austrian ministry of health (bmfg- / -iv/b/ / ). for the construction of a bipartite infectious clone, six contiguous cdna fragments encoding the genome of the lineage i wnv strain ny were chemically synthesized and integrated in bacterial expression plasmids (see section ) according to the cloning strategy outlined in fig. . three silent marker mutations were introduced (see also [ ] ) allowing the discrimination of the synthetic virus from the corresponding wild-type isolate (see table ). the six synthetically generated wnv subfragments were ligated stepwise, resulting in two plasmids with corresponding parts of the complete genomic wnv sequence. for this purpose, either unique restriction sites in the wnv sequence were used, or -where appropriate -asymmetric restriction sites were generated in the plasmid vector backbone adjacent to the wnv fragments. cleavage of these asymmetric sites created overhangs in the wnv sequence by which corresponding fragments could be fused together. following this strategy, two plasmids were generated, containing either the third (nt - under control of a t promoter) or the two-thirds (nt - , ) of the wnv genomic sequence, designated as pwnvsyn- tl or pwnvsyn- tl, respectively. each of the cloning steps was evaluated by complete sequencing of the cdna insert and no undesired sequence alterations were observed. further, in the final two plasmids no nucleotide alterations were found with the exception of the intended silent marker mutations. to analyze the functionality of the cdna system, rna transcripts corresponding to the entire genome of wnv were generated. full-length wnv cdna templates were obtained by ligation of pwnvsyn- tl and pwnvsyn- tl. genome length viral rna was transcribed and the integrity of rna transcripts was analyzed in % agarose gels containing % formaldehyde. an rna band of approximately , nucleotides was obtained, indicating the presence of wnv full-length rna (data not shown). to characterize the ability of the transcribed rna to replicate and to be translated after introduction in host cells, viral protein expression was examined by immunofluorescence (if) staining. the wnvsyn rna was electroporated into vero cells which were subjected to indirect if staining days later (fig. ) . viral protein expression was monitored with a specific polyclonal mouse anti-wnv antibody and a fitc-conjugated second antibody (see section ). cells infected with moi . of wnvwt and were used as staining control. wnvsyn-transfected and wnvwt-infected vero cells exhibited wnv protein expression in approximately % of all cells. as expected, viral antigen staining is mainly confined to perinuclear regions of the cells (fig. ) . immunofluorescence staining is only detectable from replication-and translation-competent viral templates and could not be shown in replication-deficient mutant viral genomes [ , ] thus proving the replication and protein expression capacity of the synthetic wnv genome. in order to further analyze the genotypic and phenotypic properties, a stock of the synthetic wnv was produced. confluent vero cells were transfected as described above and upon onset of cytopathic effect (cpe) after days, cell culture medium was harvested and the virus titer was determined on vero cells, yielding a titer of . × tcid /ml. overlapping dna fragments which cover the whole wnvsyn genomic coding region were amplified by pcr after cdna transcription of isolated viral rna. sequencing confirmed that the rescued viral material contained no mutations compared to the in silico designed wnv genome and the presence of the engineered nucleotide changes proved the identity of the synthetic virus. in addition, in order to show if staining behavior in vero cells not only after transfection of rna, cells were infected with moi . of wnvsyn and processed for if as described above. as expected, the wnvsyn virus stock gave rise to a similar staining pattern as seen for the wnvwt stock (fig. d) . in order to analyze the growth properties of wnvsyn and wnvwt, one step growth curves were carried out. susceptible mammalian (vero) and mosquito (c / ) cells were infected with a moi of . . viral titers, determined at the time points indicated in fig. , demonstrate that in both cell types the growth kinetics of wnvsyn match exactly those of the wild-type virus. in addition, plaque morphology ( fig. a and b) and cpe (not shown) were comparable to the wild-type control. virus grown in vero cells peaked at h posttransfection but declined from day on correlating with the onset of cpe of the infected cells from day on (fig. c) . growth kinetics in the mosquito cells was delayed as observed by others [ , ] , reaching equal titers compared to vero cells at day postinfection (fig. d) . taken together, these data indicate that wnvsyn and the corresponding wnvwt isolate are indistinguishable with respect to replication and infectivity in both tested cell lines. in addition, virulence of wnvsyn and wnvwt were compared in cohorts of -week-old balb/c mice. for this purpose mice were infected intranasally with virus dilutions corresponding to × to × tcid per animal. survival was monitored for days postinfection and ld values were calculated. similar mortalities of infected mice induced by the two wnv viruses were observed ( table ). the lethal dose for wnvsyn and wnvwt was . and table virulence of wnvsyn and wnvwt after intranasal application in balb/c mice. . log tcid , respectively. the experiment was repeated once and similar results were obtained. following the demonstration that wnvsyn exhibits indistinguishable biological properties compared to the wnv wild-type isolate, the protective efficacy of experimental vaccines derived from both viruses was analyzed. for this purpose, groups of ten mice were immunized twice with decreasing doses of formalininactivated, alum-adjuvanted whole virus vaccines derived from the viruses (see section ). quantification by elisa of vaccine preparations prior to formulation and adjuvantation confirmed the presence of equal amounts of antigen in the respective dosage groups. further, western blotting confirmed equivalent amounts and protein patterns in the two antigen preparations (fig. b) . the predominant band in these preparations is the envelope antigen (e) migrating in the kda range, the fainter bands representing the pre-membrane (prm) and the dimeric membrane (m) proteins (see also [ ] ). two weeks after the second vaccination wnv-specific neutralizing antibodies were determined by a microneutralization assay. serum analysis demonstrated high neutralizing antibody levels in both vaccine preparations (see fig. a and table ). mice were then challenged intranasally with a lethal dose ( × tcid ) of wnv wild-type virus. vaccination with both preparations resulted in a high degree of protection in vaccinated mice. complete protection was achieved using doses as low as nanograms of the wnv antigens while % of the non-vaccinated controls died. the vaccines clearly induced a dose-dependent protection correlating with nt titers (table ) . reverse genetics systems of positive-sense rna viruses allow, for instance, for mutagenesis procedures and generation of chimeric viruses and thus are invaluable tools for live vaccine development and for studying the biology of those viruses (see e.g. refs. [ , ] ). usually the starting material for the generation of seed viruses for vaccines or such reverse genetics systems are virus stocks derived from a biological source. this virus is plaque-purified, viral rna is isolated, transcribed into cdna which is amplified by pcr and in turn gets assembled into a dna copy of the viral genome. these approaches bear the risk of introducing mutations selected via plaque purification steps. to minimize this type of mutations we chose to generate a reverse genetics system using a different approach, independent of preformed viral rna components and animal sources. the feasibility of generating such systems by chemical synthesis of dna was proven previously, for instance, by the generation of poliovirus [ ] , bacteriophage x [ ] or h n spanish influenza virus [ ] , and sars-like coronavirus [ ] . on the basis of these studies, we report for the first time the generation of an , nucleotide long synthetic genome of a member of the family flaviviridae. sequence data from genbank referring to lineage i west nile virus strain ny were used as template for in silico design of the cloning strategy. rna viruses replicate their genome with an error prone mechanism (for reviews see [ ] ), resulting in a multitude of distinct but related nucleic acids forming a quasispecies [ ] . sequencing of a virus genome (usually cloned by plaque purifications prior to sequence analysis) consisting of millions of molecules, results in a 'consensus' sequence, representing the majority genotype having defined biological properties. biological properties may change, for instance, when pressure imposed by the host selects for changes of the genomic sequence, visible as a new 'consensus sequence' in the sequence analysis. in all of the cloning and propagation steps no mutations changing the wild-type consensus sequence were introduced by pcr using synthetic templates of verified nucleotide sequence proving the accuracy of this approach. thus the synthetic progeny virus was biologically indistinguishable from its natural parent. experimental inactivated vaccines derived from wnvwt and wnvsyn were highly immunogenic in animals. both vaccine preparations induced comparable levels of neutralizing antibodies and led to similar protection results. only in the low dosing groups of the protection study differences were observed that can be explained by the experimental conditions and the inherent inaccuracies of the biological system rather than by genetic differences in the two viruses. in addition, both virus stocks were indistinguishable concerning their virulence in mice. progress in synthetic biology raises biosecurity concerns. the possibility to synthesize pathogens without need for natural sources, for instance the viruses on the select agents list [ ] , results in the expansion of the potential availability of select agents (defined as biological agents and toxins regulated by the us select agent rules that have the potential to pose a severe threat to public, animal or plant health). the us government has developed guidance that addresses this issue [ ] . wnv -although a biosafety level virus -is endemic in large parts of the world and does not belong to the select agents. however, flaviviruses belonging to the tick-borne encephalitis virus complex are on this list. construction of infectious flaviviruses, involving dna synthesis, cloning, assembly into larger units, in vitro transcription and transfection steps, is a complex task and can be done in a professional environment only. a recent review on synthetic viruses discusses the dual use concerns in more detail [ ] . for vaccine manufacturing, the most important advantage of using primary seed virus stocks derived by gene synthesis is the exclusion of potential contamination with unknown and known adventitious agents -including the transmissible spongiforme encephalopathy agents -which maybe co-isolated from animalderived viruses or their host cells. furthermore, this approach renders passaging, plaque purifications and other steps to achieve satisfactory purity of seed viruses from animal sources unnecessary. our study demonstrates the feasibility of generating the flavivirus wnv in a completely synthetic approach. synthetic biology is therefore a valuable alternative to obtain viral seed stocks free from the adventitious agents that might accompany recovery from vertebrate or insect cells. emerging flaviviruses: the spread and resurgence of japanese encephalitis. west nile and dengue viruses flaviviridae: the viruses and their replication functional cdna clones of the flaviviridae: strategies and applications a stable full-length yellow fever virus cdna clone and the role of conserved rna elements in flavivirus replication infectious cdna clones of langat tick-borne flavivirus that differ from their parent in peripheral neurovirulence identification of a major determinant of mouse neurovirulence of dengue virus type using stably cloned 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infectious cdna clone of attenuated langat tick-borne flavivirus (strain e ) and a deletion mutant constructed from it exhibit decreased neuroinvasiveness in immunodeficient mice infectious rna transcripts from fulllength dengue virus type cdna clones made in yeast construction of a full length infectious clone for dengue- virus western pacific, strain transcription of infectious yellow fever rna from full-length cdna templates produced by in vitro ligation infectious cdna clone of the epidemic west nile virus from new york city infectious japanese encephalitis virus rna can be synthesized from in vitro-ligated cdna templates an infectious clone of the west nile flavivirus a new strategy in design of +rna virus infectious clones enabling their stable propagation in e. coli chemical synthesis of poliovirus cdna: generation of infectious virus in the absence of natural template synthetic viruses: a new opportunity to understand and prevent viral disease functional analysis of the tick-borne encephalitis virus cyclization elements indicates major differences between mosquito-borne and tick-borne flaviviruses proteolytic activation of tick-borne encephalitis virus by furin kunjin virus replicons: an rnabased, non-cytopathic viral vector system for protein production, vaccine and gene therapy applications preclinical and clinical development of yfv d-based chimeric vaccines against dengue, west nile and japanese encephalitis viruses the test-tube synthesis of a chemical called poliovirus generating a synthetic genome by whole genome assembly: phix bacteriophage from synthetic oligonucleotides characterization of the reconstructed spanish influenza pandemic virus synthetic recombinant bat sars-like coronavirus is infectious in cultured cells and in mice rates of spontaneous mutation viral quasispecies addressing biosecurity concerns related to the synthesis of select agents. nih we thank helga savidis-dacho and her team for performing the animal experiments, kathrin janecki, marie-luise zips and petra cech for expert technical assistance and the geneart team for providing the cloning strategy and the six genomic plasmids. key: cord- -ywaefpe authors: rodon, jordi; muñoz-basagoiti, jordana; perez-zsolt, daniel; noguera-julian, marc; paredes, roger; mateu, lourdes; quiñones, carles; erkizia, itziar; blanco, ignacio; valencia, alfonso; guallar, víctor; carrillo, jorge; blanco, julià; segalés, joaquim; clotet, bonaventura; vergara-alert, júlia; izquierdo-useros, nuria title: pre-clinical search of sars-cov- inhibitors and their combinations in approved drugs to tackle covid- pandemic date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: ywaefpe there is an urgent need to identify novel drugs against the new coronavirus. although different antivirals are given for the clinical management of sars-cov- infection, their efficacy is still under evaluation. here, we have screened existing drugs approved for human use in a variety of diseases, to compare how they counteract sars-cov- -induced cytopathic effect and viral replication in vitro. among the potential antivirals tested herein that were previously proposed to inhibit sars-cov- infection, only % had in vitro antiviral activity. moreover, only eight families had an ic below µm or iu/ml. these include chloroquine derivatives and remdesivir, along with plitidepsin, cathepsin inhibitors, nelfinavir mesylate hydrate, interferon -alpha, interferon-gamma, fenofibrate and camostat. plitidepsin was the only clinically approved drug displaying nanomolar efficacy. four of these families, including novel cathepsin inhibitors, blocked viral entry in a cell-type specific manner. since the most effective antivirals usually combine therapies that tackle the virus at different steps of infection, we also assessed several drug combinations. although no particular synergy was found, inhibitory combinations did not reduce their antiviral activity. thus, these combinations could decrease the potential emergence of resistant viruses. antivirals prioritized herein identify novel compounds and their mode of action, while independently replicating the activity of a reduced proportion of drugs which are mostly approved for clinical use. combinations of these drugs should be tested in animal models to inform the design of fast track clinical trials. there is an urgent need to identify novel drugs against the new coronavirus. although different antivirals are given for the clinical management of sars-cov- infection, their efficacy is still under evaluation. here, we have screened existing drugs approved for human use in a variety of diseases, to compare how they counteract sars-cov- -induced cytopathic effect and viral replication in vitro. among the potential antivirals tested herein that were previously proposed to inhibit sars-cov- infection, only % had in vitro antiviral activity. moreover, only eight families had an ic below µm or iu/ml. these include chloroquine derivatives and remdesivir, along with plitidepsin, cathepsin inhibitors, nelfinavir mesylate hydrate, interferon -alpha, interferon-gamma, fenofibrate and camostat. plitidepsin was the only clinically approved drug displaying nanomolar efficacy. four of these families, including novel cathepsin inhibitors, blocked viral entry in a cell-type specific manner. since the most effective antivirals usually combine therapies that tackle the virus at different steps of infection, we also assessed several drug combinations. although no particular synergy was found, inhibitory combinations did not reduce their antiviral activity. thus, these combinations could decrease the potential emergence of resistant viruses. antivirals prioritized herein identify novel compounds and their mode of action, while independently replicating the activity of a reduced proportion of drugs which are mostly approved for clinical use. combinations of these drugs should be tested in animal models to inform the design of fast track clinical trials. a novel betacoronavirus, the severe acute respiratory syndrome coronavirus (sars-cov- ), is causing a respiratory disease pandemic that began in wuhan, china, in november , and has now spread across the world (chen et al., ) . to date, remdesivir is the only approved antiviral drug for the specific treatment of this coronavirus infectious disease or covid- (beigel et al., ; grein et al., ) . however, several drugs are being used in the frontline of clinical management of sars-cov- -infected individuals in hospitals all around the world, to try to avoid the development of the covid- associated pneumonia, which can be fatal. by the end of september , almost a million people had died from covid- , and over million people have been infected (who situation report). although different drug regimens are being applied to hospitalized patients, no clinical study has evidenced their efficacy yet. under this scenario, initiatives launched by the world health organization (who), such as the solidarity study that has compared remdesivir, hydroxychloroquine, ritonavir/lopinavir and ritonavir/lopinavir plus ßinterferon regimes, have been of critical importance to prioritize the use of the most active compounds (who, ) . unfortunately, although remdesivir has proven efficacy in randomized controlled trials (beigel et al., ; grein et al., ) , a recent update of the who clinical trial has failed to detect any effect on overall mortality, initiation of ventilation and duration of hospital stay with any of the antivirals tested (pan et al., ) . thus, there is an urgent need to identify novel therapeutic approaches for individuals with covid- developing severe disease and fatal outcomes. in this report we present a prioritized list of effective compounds with proven antiviral efficacy in vitro to halt sars-cov- replication. compounds were analyzed depending on their expected mechanism of action, to identify candidates tackling diverse steps of the viral life cycle. sars-cov- entry requires viral binding and spike protein activation via interaction with the cellular receptor ace and the cellular protease tmprss (hoffmann et al., ) , a mechanism favored by viral internalization via endocytosis. interference with either of these processes has proven to decrease sars-cov- infectivity (hoffmann et al., ; monteil et al. ) , and therefore, inhibitors targeting viral entry may prove valuable. in addition, sars-cov- enters into the cells via endocytosis and accumulates in endosomes where cellular cathepsins can also prime the spike protein and favor viral fusion upon cleavage (hoffmann et al., ; mingo et al., ; simmons et al., ) , providing additional targets for antiviral activity. once sars-cov- fuses with cellular membranes, it triggers viral rna release into the cytoplasm, where polyproteins are translated and cleaved by proteases (song et al., ) . this leads to the formation of an rna replicase-transcriptase complex driving the production of negative-stranded rna via both replication and transcription (song et al., ) . negative-stranded rna transcribes into positive rna genomes, allowing for the translation of viral nucleoproteins, which assemble in viral capsids at the cytoplasm (song et al., ) . these capsids then bud into the lumen of endoplasmic reticulum (er)-golgi compartments, where viruses are finally released into the extracellular space by exocytosis. potentially, any of these viral cycle steps could be targeted with antivirals, so we have thus searched for these compounds as well. finally, as the most effective antiviral treatments are usually based on combined therapies that tackle distinct steps of the viral life cycle, we also tested the active compounds in combination. these combinations may be critical to abrogate the potential emergence of resistant viruses and to increase antiviral activity, enhancing the chances to improve clinical outcome. we have tested the antiviral activity of different clinically available compounds and their combinations by assessing their ability to inhibit viral induced cytopathic effect in vitro. our strategy was circumscribed mostly to compounds approved for clinical use, since they are ideal candidates for entering into fast track clinical trials. drug selection criteria first focused on compounds already being tested in clinical trials, along with well-known human immunodeficiency virus- (hiv- ) and hepatitis c virus (hcv) protease inhibitors, as well as other compounds suggested to have potential activity against sars-cov- in molecular docking analysis or in vitro assays. we first assessed the activity of compounds with hypothetical capacity to inhibit viral entry, and then we focused on drugs thought to block viral replication upon sars-cov- fusion. molecular docking studies provided an additional candidates, which were predicted to inhibit the sars-cov- main protease. finally, compounds with unknown mechanism of action were also assessed. by these means, we have compared drugs and of their combinations for their capacity to counteract sars-cov- induced cytopathic effect in vitro. we first tested compounds that could have an effect before viral entry by impairing viruscell fusion (supp . table ) . hydroxychloroquine is an anti-malarial drug that exerts its activity by disrupting the endosome pathway, and that has been proposed as an anti-sars-cov- agent wang et al., ) . we confirmed the inhibitory effect of hydroxychloroquine against sars-cov- -induced cellular cytotoxicity on vero e cells . a constant concentration of a clinical isolate of sars-cov- (id epi_isl_ ) was mixed with increasing concentrations of hydroxychloroquine and added to vero e cells. to control for drug-induced cytotoxicity, vero e were also cultured with increasing concentrations of hydroxychloroquine in the absence of sars-cov- . by these means we calculated the concentration at which hydroxychloroquine achieved a % maximal inhibitory capacity (ic ). as shown in fig. a , this drug was able to inhibit viral-induced cytopathic effects at concentrations where no cytotoxic effects of the drug were observed. the mean ic value of this drug in repeated experiments was always below µm (supp . table ) . these results aligned with previous reports highlighting the in vitro inhibitory capacity of chloroquine derivatives wang et al., ) , but contrasted with data on animal models (maisonnasse et al., ; rosenke et al., ) or currently ongoing clinical trials that have failed to detect any associated benefit of hydroxychloroquine treatment (boulware et al., ; cavalcanti et al., ) . since hydroxychloroquine was first administered in combination with the antibiotic azithromycin (gautret et al., ) , which induces antiviral responses in bronchial epithelial cells (gielen et al., ) , we further tested the activity of this compound in our assay. however, in the vero e model, azithromycin did not show any antiviral effect (fig. a) , and the combination of hydroxychloroquine with azithromycin had a similar activity to that of the chloroquine derivative alone (fig. a) . indeed, this was also the case when we tested hydroxychloroquine in combination with different hiv- protease inhibitors and other relevant compounds currently being tested in clinical trials (supp. table ). additional food and drug administration (fda)-approved compounds previously used to abrogate viral entry via clathrin-mediated endocytosis were also tested in this sars-cov- -induced cytotoxicity assay (supp . table ) . indeed, interference with clathrinmediated endocytosis is one of the potential mechanisms by which hydroxychloroquine may exert its therapeutic effect against sars-cov- . one of these compounds was amantadine, which blocks coated pit invaginations at the plasma membrane (phonphok and rosenthal, ) and is licensed against influenza a virus infections and as a treatment for parkinson's disease. in addition, we also tested chlorpromazine, an antipsychotic drug that inhibits clathrin-mediated endocytosis by preventing the assembly and disassembly of clathrin networks on cellular membranes or endosomes (wang et al., ) . when we assessed the antiviral efficacy of these clathrin inhibitors against sars-cov- , we did not find any prominent effect; only a partial inhibition at µm for amantadine (fig. b) . the broad cathepsin b/l inhibitor e -d also showed partial inhibitory activity (fig. b) . e -d exerts activity against viruses cleaved by cellular cathepsins upon endosomal internalization, as previously described using pseudotyped sars-cov- (hoffmann et al., ) . while these results could not be confirmed using the specific cathepsin b inhibitor ca- -me due to drug-associated toxicity (supp . table ) , it is important to highlight that none of these cathepsin inhibitors is approved for clinical use. these data suggest that sars-cov- entry in vero e partially relies on clathrin-mediated endocytosis and cellular cathepsins, which cleave the viral spike protein and allow viral fusion once sars-cov- is internalized in endosomes. however, hydroxychloroquine antiviral activity was much more potent than that exerted by amantadine or e- d ( fig. a-b) . recently, it has also been suggested that hydroxychloroquine could block sars-cov- spike interaction with gm gangliosides (fantini et al., ) . gm gangliosides are enriched in cholesterol domains of the plasma membrane and have been previously shown to bind to sars-cov spike protein (lu et al., ) . this mode of viral interaction is aligned with the capacity of methyl-beta cyclodextrin, which depletes cholesterol from the plasma membrane to abrogate sars-cov- induced cytopathic effect (fig. b) , as previously reported for sars-cov (lu et al., ) . removal of cholesterol redirected ace receptor to other domains, but did not alter the expression of the viral receptor (lu et al., ) . moreover, nb-dnj, an inhibitor of ganglioside biosynthesis pathway, also decreased sars-cov- cytopathic effect (fig. b) . these results highlight the possible role of gangliosides in viral binding, although the polar head group of gm ganglioside ( ' sialyllactose) was not able to reduce viral-induced cytopathic effect (supp. table ). agents involved in autophagy, such as the becn -stabilizing compounds niclosamide or ciclesonide, inhibit the release of infectious sars-cov- to the supernatant (gassen et al., ) or reduce the expression of viral nucleoprotein h post-infection (jeon et al., ) . however, these autophagy inhibitors were highly toxic in our three-day assay (supp . table ). arbidol is a compound that intercalates into membrane lipids leading to the inhibition of membrane fusion between viruses and cells, and between viruses and endosomal membranes (haviernik et al., ) . although arbidol has exhibited in vitro efficacy against sars-cov- (li, ) it showed drug associated cytotoxicity in our assay (supp. table ). we also tested the antiviral activity of two jak inhibitors: baricitinib and tofacitinib. baricitinib was previously suggested to reduce viral entry by interfering with ap -associated protein kinase (aak ) necessary for clathrin mediated endocytosis stebbing et al., ) . however, neither this compound or tofacitinib protected vero e cells from sars-cov- induced cytopathic effect (supp. table ). while we did not detect an antiviral effect for jak inhibitors, these compounds may still be useful to control hyperinflammation and cytokine storm at later stages of infection ). finally, we tested camostat, a serine protease inhibitor with capacity to abrogate sars-cov- spike priming on the plasma membrane of human pulmonary cells and avoid viral fusion (hoffmann et al., ) . camostat showed no antiviral effect on vero e cells (fig. c) , what indicates that the alternative viral endocytic route is the most prominent entry route in this renal cell type. of note, a broader cellular protease inhibitor such as the alfa -antitrypsin (att), used to treat severe att human deficiency, was able to exert an antiviral effect on vero e . however, it required high concentrations that will most likely rely on the activity of these proteases in the endosomal route (fig. c) . in order to confirm that previously identified compounds listed in supp. table specifically inhibit the viral entry step, we next employed a luciferase-based assay using pseudotyped lentivirus expressing the spike protein of sars-cov- , which allows to detect viral fusion on hek- t cells transfected with ace . as a control, we used the same lentiviruses pseudotyped with a vsv glycoprotein, where no entry inhibition above % was detected for any of the drugs tested (data not shown). in sharp contrast, sars-cov- pseudoviruses were effectively blocked by most of the drugs previously tested on vero e with wild-type virus (fig. d) . the main differences were observed with ca- -me, ciclesonide and arbidol. these compounds showed a partial blocking effect on ace hek- t cells that was not obvious when using replication competent sars-cov- on vero e (supp. fig. ). in addition, nb-dnj failed to block viral entry ( fig. d ), suggesting that ganglioside dependence may be reduced in ace overexpressing cells or, alternatively, that this drug requires a longer exposure time to effectively reduce the content of gangliosides via biosynthesis blocking. overall, using alternative sars-cov- viral systems, we could identify chloroquine derivatives, cathepsin inhibitors and cholesterol depleting agents as the most promising candidates to block sars-cov- endocytosis in vero e and hek- t cells transfected with ace . however, chloroquine derivatives were the only ones that displayed an ic below µm (supp . table ) , and were also active abrogating pseudoviral entry into hek- t cells expressing ace (fig. e) . although camostat failed to inhibit viral fusion on ace hek- t cells (fig. e) , its activity was rescued when these cells were transfected with tmprss . the opposite effect was observed for chloroquine, which reduced its inhibitory activity on tmprss transfected cells (fig. e ). thus, the expression of cellular proteases on the plasma membrane facilitates the fusion with viral membranes, decreasing the likelihood of viral entry through the endosomal route. these data concur with previous findings in pulmonary cells, where viral entry via endosomal route was not active since chloroquine failed to abrogate viral fusion (maisonnasse et al., ) ; however, camostat effectively blocked this entry (hoffmann et al., ) . our results highlight that alternative routes govern sars-cov- viral entry and these pathways vary depending on the cellular target. thus, effective treatments may need to block both plasma membrane fusion and endosomal routes to fully achieve viral suppression. in our search for antivirals inhibiting post-viral entry steps, we first focused on remdesivir, which has in vitro activity against sars-cov- after viral entry and has already been approved for the treatment of covid- by the fda and ema. we further confirmed the in vitro capacity of remdesivir to inhibit sars-cov- induced cytopathic effect on vero e ( fig. a) . the mean ic value of this drug in repeated experiments was always below µm (supp. table ). in combination with hydroxychloroquine, however, remdesivir did not significantly modified its own antiviral effect ( fig. b) , either when hydroxychloroquine was added at increasing concentrations or at different fixed concentrations of the drug. this was also the case for other antivirals tested in combination (supp. table ). of note, other rna polymerase inhibitors such as galdesivir, which was proposed to tightly bind to sars-cov- rna-dependent rna polymerase (elfiky, ) , showed no antiviral effect (supp . table ). favipiravir, approved by the national medical products administration of china as the first anti-covid- drug in china (tu et al., ) , showed only partial inhibitory activity at the non-toxic concentration of µm (supp . table ) . we also assessed clinically approved protease inhibitors with potent activity against hiv- . however, none of the hiv- protease inhibitors detailed in supp. table showed remarkable protective antiviral activity against sars-cov- infection on vero e cells, with the exception of nelfinavir mesylate hydrate, which showed an ic value below µm (supp . table and fig. c ). lopinavir and tipranavir inhibited sars-cov- induced cytopathic effect at the non-toxic concentration of µm, and amprenavir exhibited activity at the non-toxic concentration of µm (fig. c) . darunavir, which is currently being tested in ongoing clinical trials, showed partial inhibitory activity at µm, although this concentration had . ± . % of cytotoxicity associated (fig. c) . of note, we tested hiv- reverse transcriptase inhibitors such as tenofovir disoproxil fumarate, emtricitabin, tenofovir alafenamide, and their combinations, but they also failed to show any antiviral effect against sars-cov- (supp. fig. ) . these results indicate that future clinical trials should contemplate the limited antiviral effect displayed by these anti-hiv- inhibitors against sars-cov- in vitro. we also assessed the inhibitory capacity of hcv protease inhibitors, but none showed any antiviral activity (supp. table ). of note, exogenous interferons alpha and gamma displayed antiviral activity against sars-cov- (supp. table ). in light of these results, we tested the inhibitory effect of the tlr agonist vesatolimod that triggers interferon production. although this agonist was not able to protect from the viralinduced cytopathic effect on vero e (supp . table ) , as expected since it is an interferon-producer deficient cell line (emeny and morgan, ) , it could still be useful in other competent cellular targets. since severe covid- patients display impaired interferon responses (hadjadj et al., ) , these strategies may be valuable to avoid disease complication. in addition, we also assessed several compounds with the best computational docking scores among approved drugs against the cl protease of sars-cov- , but none of them were effective to protect vero e from viral induced cytopathic effect (supp. table ) . the most potent antiviral tested was plitidepsin (fig. d) , which targets the eukaryotic elongation factor a (eef a ) and has been previously used for the treatment of multiple myeloma. the mean ic value of this drug in repeated experiments was always in nm concentrations (supp . table ). in combination with other active antivirals, we did not observe a reduction on ic values (supp. table ). this result indicates no significant synergy, but also highlights the possibility of using plitidepsin without reducing its antiviral activity in combined therapies (fig. d) , what could be relevant to avoid possible selection of resistant viruses. overall, plitidepsin showed the lowest ic values of all the compounds tested in this in vitro screening ( table ) . we also assessed the inhibitory capacity of several inhibitors and broad anti-bacterial, anti-parasitic, anti-malarial, anti-influenza and anti-fungal compounds, along with other pharmacological agents previously suggested to interfere with sars-cov- infection (supp . table ). such was the case of ivermectin, an fda-approved broad spectrum anti-parasitic agent previously reported to inhibit the replication of sars-cov- in vitro as measured by rna accumulation (caly et al., ) . however, among these potential antivirals, only three types of molecules exerted detectable antiviral activity in our assay: itraconazole, fenofibrate, and calpain and cathepsin inhibitors such as mdl and npo compounds. itraconazole, an antifungal that may interfere with internal sars-cov- budding within infected cells (wu et al., ) , displayed an ic value of µm ( fig. a and supp. table ). fenofibrate is clinically used to treat dyslipidemia via activation of ppara, and also inhibited the cytopathic effect exerted by sars-cov- on vero e at µm ( fig. b and supp. table ). as fenofibrate is a regulator of cellular lipid metabolism, we made use of the luciferase-based viral entry assay to try to elucidate its mode of action. when lentiviruses pseudotyped with the spike protein of sars-cov- were added to ace- expressing hek- t cells in the presence of fenofibrate, viral entry was abrogated (fig. c) . the most potent agent found was mdl , a calpain iii inhibitor in a pre-clinical stage of development that displayed activity in the nanomolar range ( fig. d and supp. (riva et al., ) . moreover, three out of four different calpain and cathepsin inhibitors named npo showed potent antiviral activity too (supp. figure ) . of note, in combination with other active antivirals, we did not observe a reduction on ic values of mdl (supp. table ) . inhibitors of calpains, which are cysteine proteases, might impair the activity of viral proteases like cl (main protease) and plpro (papain-like protease) (riva et al., ; schneider et al., ) . however, calpain inhibitors may also inhibit cathepsin bmediated processing of viral spike proteins or glycoproteins, including sars-cov and ebola (schneider et al., ; zhou and simmons, ) . to understand the mechanisms of action of calpain and cathepsin inhibitors such as mdl , we added lentiviruses pseudotyped with the spike protein of sars-cov- to ace- -expressing hek- t cells and the same cells also expressing tmprss in the presence of this drug. importantly, mdl only blocked viral entry in ace- -expressing cells (fig. e) . this result indicates that mdl blocks cathepsins that are implicated in sars-cov- entry via the alternative endosomal pathway, as described for chloroquine derivatives and e- d (fig. e) , which are all active when tmprss is not present and their inhibitor camostat displays no activity (fig. e) . in conclusion, among the compounds and their combinations tested herein for their potential capacity to abrogate sars-cov- cytopathic effect, we only found compounds with antiviral activity, and only eight types of these drugs had an ic below µm or iu/ml ( table ) . these eight families of compounds were able to abrogate sars-cov- release to the supernatant in a dose dependent manner (fig. ) , indicating that the reduction in the cytopathic effect that we had measured in cells correlates with viral production. as these eight families of compounds tackle different steps of the viral life cycle, they could be tested in combined therapies to abrogate the potential emergence of resistant viruses. we have assessed the anti-sars-cov- activity of clinically approved compounds that may exert antiviral effect alone or in combination. although we were not able to detect any remarkable synergy in vitro, combined therapies are key to tackle viral infections and to reduce the appearance of viral resistance. we have tested more than seventy compounds and their combinations, and verified a potent antiviral effect of hydroxychloroquine and remdesivir, along with plitidepsin, cathepsin and calpain inhibitors mdl and npo, nelfinavir mesylate hydrate, interferon a, interferon-g and fenofibrate. these are therefore the most promising agents found herein that were able to protect cells from viral-induced cytopathic effect by preventing viral replication. our findings highlight the utility of using hydroxychloroquine and mdl or other cathepsin inhibitors to block viral entry via the endosomal pathway in kidney cell lines such as vero e or hek- t. however, the endosomal viral entry route is absent in pulmonary cells and, therefore, camostat should be considered as the primary inhibitor to limit sars-cov- entry in pulmonary tissues or in cells expressing tmprss . these findings can explain why randomized clinical trials using hydroxychloroquine have failed to show a significant protective effect (boulware et al., ; cavalcanti et al., ) . nonetheless, in combined therapies, it should be noted that agents targeting the alternative endosomal sars-cov- entry route such as hydroxychloroquine or mdl could be key to stop viral dissemination in other extrapulmonary tissues where viral replication has been already detected , and viral entry could take place through this endosomal pathway. this could partially explain why in a retrospective observational study including more than patients, hydroxychloroquine treatment showed a significant reduction of in-hospital mortality (arshad et al., ) . thus, since alternative routes govern sars-cov- viral entry depending on the cellular target (ou et al., a) , effective treatments might be needed to block both plasma membrane fusion and endosomal entry to broadly achieve viral suppression. sars-cov- replication could be effectively blocked using nelfinavir mesylate hydrate, remdesivir and plitidepsin. while nelfinavir showed lower potency, remdesivir and plitidepsin were the most potent agents identified. however, remdesivir and plitidepsin are not yet suitable for oral delivery and require intravenous injection, complicating their clinical use for prophylaxis. finally, we also confirmed the antiviral effect of type i and ii interferons as well as fenofibrate, which have been extensively used in the clinic for many years and may therefore prove valuable for therapeutic use. the data presented herein should be interpreted with caution, as the ic values of drugs obtained in vitro may not reflect what could happen in vivo upon sars-cov- infection. the best antiviral compounds found in the present study need to be tested in adequate animal models. this strategy already helped to confirm the activity of remdesivir against sars-cov- , while also questioning the use of hydroxychloroquine in monotherapy (maisonnasse et al., ) . thus, assessing antiviral activity and safety in animal models is key to identify and advance those compounds with the highest potential to succeed in upcoming clinical trials. in turn, in vitro results confirmed in animal models will provide a rational basis to perform future clinical trials not only for treatment of sars-cov- -infected individuals, but also for pre-exposure prophylaxis strategies that could avoid novel infections. prophylaxis could be envisioned at a population level or to protect the most vulnerable groups, and should be implemented until an effective vaccine is developed. in particular, orally available compounds with proven safety profiles, such as fenofibrate, could represent promising agents. germans trias i pujol (hugtip) approved this study. the individual who provided the sample to isolate virus gave a written informed consent to participate. eagle medium, (dmem; lonza) supplemented with % fetal calf serum (fcs; euroclone), u/ml penicillin, µg/ml streptomycin, and mm glutamine (all thermofisher scientific). hek- t (atcc repository) were maintained in dmem with % fetal bovine serum, iu/ml penicillin and µg/ml streptomycin (all from invitrogen). hek- t overexpressing the human ace were kindly provided by integral molecular company and maintained in dmem (invitrogen) with % fetal bovine serum, iu/ml penicillin and µg/ml streptomycin, and µg/ml of puromycin (all from invitrogen). tmprss human plasmid (origene) was transfected using x-tremegene hp transfection reagent (merck) on hek- t overexpressing the human ace and maintained in the previously described media containing mg/ml of geneticin (invitrogen) to obtain tmprss /ace hek- t cells. virus isolation, titration and sequencing. sars-cov- was isolated from a nasopharyngeal swab collected from an -year-old male patient giving informed consent and treated with betaferon and hydroxychloroquine for days before sample collection. the swab was collected in ml medium (deltaswab vicum) to reduce viscosity and stored at - ºc until use. vero e cells were cultured on a cell culture flask ( cm ) at . x cells overnight prior to inoculation with ml of the processed sample, for h at ºc and % co . afterwards, ml of % fcs-supplemented dmem were supplied and cells were incubated for h. supernatant was harvested, centrifuged at x g for min to remove cell debris and stored at - ºc. cells were assessed daily for cytopathic effect and the supernatant was subjected to viral rna extraction and specific rt-qpcr using the sars-cov- upe, rdrp and n assays (corman et al., ) . the virus was propagated for two passages and a virus stock was prepared collecting the supernatant from vero e . viral rna was extracted directly from the virus stock using the indimag pathogen kit (indical biosciences) and transcribed to cdna using the primescript™ rt reagent kit (takara) using oligo-dt and random hexamers, according to the manufacturer's protocol. dna library preparation was performed using swift amplicon sars-cov- panel (swift biosciences). sequencing ready libraries where then loaded onto illumina miseq platform and a bp paired-end sequencing kit. sequence reads were quality filtered and adapter primer sequences were trimmed using trimmomatic. amplification primer sequences were removed using cutadapt (martin, ) . sequencing reads were then mapped against coronavirus reference (nc_ . ) using bowtie tool (langmead, b. and salzberg, s, ) . consensus genomic sequence was called from the resulting alignment at a x x average coverage using samtools (li et al., ) . genomic sequence was deposited at gisaid repository (http://gisaid.org) with accession id epi_isl_ . pseudovirus production. hiv- reporter pseudoviruses expressing sars-cov- spike protein and luciferase were generated using two plasmids. pnl - .luc.r-.e-was obtained from the nih aids repository. sars-cov- .sctΔ was generated (geneart) from the full protein sequence of sars-cov- spike with a deletion of the last amino acids in c-terminal, human-codon optimized and inserted into pcdna . -topo (ou et al., b) . spike plasmid was transfected with x-tremegene hp transfection reagent µm to . nm at ⅕ serial dilutions. plitidepsin was also assayed at concentrations ranging from µm to . nm at / dilutions. interferons were assayed at concentrations ranging from to . iu/ml at ⅕ serial dilutions. when two drugs were combined, each one was added at a : molar ratio at concentrations ranging from µm to . nm at ⅕ serial dilutions. in combination with other drugs, plitidepsin was also assayed at concentrations ranging from µm to . nm at / dilutions. e cells together with . tcid /ml of sars-cov- , a concentration that achieves a % of cytopathic effect. untreated non-infected cells and untreated virus-infected cells were used as negative and positive controls of infection, respectively. to detect any drugassociated cytotoxic effect, vero e cells were equally cultured in the presence of increasing drug concentrations, but in the absence of virus. cytopathic or cytotoxic effects of the virus or drugs were measured days after infection, using the celltiter-glo luminescent cell viability assay (promega). luminescence was measured in a fluoroskan ascent fl luminometer (thermofisher scientific). were adjusted to a non-linear fit regression model, calculated with a four-parameter logistic curve with variable slope. cells not exposed to the virus were used as negative controls of infection, and were set as % of viability to normalize data and calculate the percentage of cytopathic effect. statistical differences from % were assessed with a one sample t test. all analyses and figures were generated with the graphpad prism v . b software. in silico drug modeling. we performed glide docking using an in-house library of all approved drug molecules on the cl protease of sars-cov- . for this, two different receptors were used, the lu pdb structure, after removing the covalently bound inhibitor, and a combination of two crystals from the diamond collection (https://www.diamond.ac.uk/covid- ). receptors were prepared with the schrodinger's protein wizard and glide sp docking was performed with two different hydrogen bond constraints: glu and his (with epsilon protonation); we enforced single constraints and also attempted the combination of both. the best molecules, based on glides's docking score were selected. top docking scores, however, did not exceed - , indicating poor potential binding. pseudovirus assay. hek- t overexpressing the human ace and tmprss were used to test antivirals at the concentrations found to be effective for sars-cov- without toxicity, which were the following: µm for niclosamide; µm for chloroquine, chlorpromazine, ciclesonide, mdl and fenofibrate; µm for hydroxychloroquine, ca- -me and arbidol hcl; µm for e- d; µm for baricitinib; µm for amantadine, nb-dnj, ' sialyl-lactose na salt, tofacitinib, and camostat mesylate; µm for methyl-b-cyclodextrin, and , mg/ml for att. a constant pseudoviral titer was used to pulse cells in the presence of the drugs. h postinoculation, cells were lysed with the glo luciferase system (promega). luminescence was measured with an ensight multimode plate reader (perkin elmer). table . compounds with antiviral activity grouped in colors depending on their ic values, expressed in µm unless otherwise indicated. activity of hydroxychloroquine and azithromycin. cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence of increasing concentrations of hydroxychloroquine, azithromycin, and their combination. drugs were used at a concentration ranging from . nm to µm. when combined, each drug was added at the same concentration. non-linear fit to a variable response curve from one representative experiment with two replicates is shown (red lines), excluding data from drug concentrations with associated toxicity. the particular ic value of this graph is indicated. cytotoxic effect on vero e cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). b. cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence of increasing concentrations of amantadine, a clathrin-mediated endocytosis inhibitor, e- d, a pancathepsin inhibitor acting downstream once viruses are internalized in endosomes, nb-dnj, an inhibitor of ganglioside biosynthesis and methyl-b-cyclodextrin, a cholesteroldepleting agent. all drugs were used at a concentration ranging from . nm to µm aside from methyl-b-cyclodextrin, which was used times more concentrated. nonlinear fit to a variable response curve from one experiment with two replicates is shown (red lines). cytotoxic effect on vero e cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). c. cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence of increasing concentrations of camostat, a tmprss inhibitor, and att, an alfa- antyitrypsin, a broad cellular protease inhibitor, as described in a. d. effect of entry inhibitors on luciferase expression of reporter lentiviruses pseudotyped with sars-cov- spike in ace expressing hek- t cells. values are normalized to luciferase expression by mock-treated cells set at %. mean and s.e.m. from two experiments with two replicates. cells were exposed to fixed amounts of sars-cov- spike lentiviruses in the presence of a non-toxic constant concentration of the drugs tested on vero e . statistical deviations from % were assessed with a one sample t test. e. comparison of entry inhibitors blocking viral endocytosis, such as chloroquine, with inhibitors blocking serine protease tmprss expressed on the cellular membrane, such as camostat, on different cell lines. ace expressing hek- t cells transfected or not with tmprss were exposed to sars-cov- spike lentiviruses as described in b. values are normalized to luciferase expression by mock-treated cells set at %. mean and s.e.m. from at least two representative experiments with two replicates. statistical deviations from % were assessed with a one sample t test. a. cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence of increasing concentrations of remdesivir. drug was used at a concentration ranging from . nm to µm. non-linear fit to a variable response curve from one representative experiment with two replicates is shown (red lines), excluding data from drug concentrations with associated toxicity. the particular ic value of this graph is indicated. cytotoxic effect on vero e cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). b. cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence of increasing concentrations of remdesivir and its combination with hydroxychloroquine, as detailed in a. drugs in combination were used at a concentration ranging from . nm to µm (left panel). alternatively, remdesivir was used at a concentration ranging from . nm to µm at the fixed indicated concentrations of hydroxychloroquine (right panel). c. cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence of increasing concentrations of protease inhibitors against hiv- . nelfinavir mesylate hydrate was the only drug with activity. inhibitors were used at a concentration ranging from . nm to µm. the particular ic value of this graph is indicated d. cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence of increasing concentrations of plitidepsin and its combinations with hydroxychloroquine and remdesivir. when combined, each drug was added at the same concentration. drugs were used at a concentration ranging from . nm to µm. the particular ic value of these graphs is indicated. cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence of increasing concentrations of itraconazole. drug was used at a concentration ranging from . nm to µm . non-linear fit to a variable response curve from one representative experiment with two replicates is shown (red lines), excluding data from drug concentrations with associated toxicity. the particular ic value of this graph is indicated. cytotoxic effect on vero e cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). b. cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence were assessed with a one sample t test. supp. table . antiviral activity of potential entry inhibitors tested against sars-cov- . na; not active. ic values are reported in µm unless otherwise indicated. supp. table . antiviral activity of potential inhibitors against sars-cov- tested in combination. na; not active. table . antiviral activity of potential post-entry inhibitors against sars-cov- . na; not active. ic values are reported in µm unless otherwise indicated. supp. table . antiviral activity of potential inhibitors against sars-cov- with predicted capacity to block sars-cov- viral protease. na; not active. supp. table . antiviral activity of potential inhibitors against sars-cov- with unknown mechanism of action. na; not active. cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence of increasing concentrations of hiv- reverse transcriptase inhibitors. drugs were used at a concentration ranging from . nm to µm. non-linear fit to a variable response curve from one experiment with two replicates is shown (red lines). cytotoxic effect on vero e cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). cytopathic effect on vero e cells exposed to a fixed concentration of sars-cov- in the presence of increasing concentrations of calpain and cathepsin inhibitors npo. drugs were used at a concentration ranging from . nm to µm. non-linear fit to a variable response curve from one experiment with two replicates is shown (red lines). cytotoxic effect on vero e cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). the research of cbig consortium (constituted by irta-cresa, bsc, & irsicaixa) is supported by grifols pharmaceutical. the authors also acknowledge the crowdfunding initiative #yomecorono (https://www.yomecorono.com). js, jva and niu have nonrestrictive funding from pharma mar to study the antiviral effect of plitidepsin. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. a patent application based on this work has been filed (ep . ). the authors declare that no other competing financial interests exist. treatment with hydroxychloroquine, azithromycin, and combination in patients hospitalized with covid- remdesivir for the treatment of covid- -preliminary report a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid- the fdaapproved drug ivermectin inhibits the replication of sars-cov- in vitro hydroxychloroquine with or without azithromycin in mild-to-moderate covid- epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study detection of novel coronavirus ( -ncov) by real-time rt-pcr ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir against sars-cov- rna dependent rna polymerase (rdrp): a molecular docking study regulation of the interferon system: evidence that vero cells have a genetic defect in interferon production structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against sars-cov- infection analysis of sars-cov- -controlled autophagy reveals spermidine hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial azithromycin induces anti-viral responses in bronchial epithelial cells compassionate use of remdesivir for patients with severe covid- impaired type i interferon activity and inflammatory responses in severe covid- patients histopathological findings and viral tropism in uk patients with severe fatal covid- : a post-mortem study arbidol (umifenovir): a broad-spectrum antiviral drug that inhibits medically important arthropod-borne flaviviruses the novel coronavirus ( -ncov) uses the sarscoronavirus receptor ace and the cellular protease tmprss for entry into target cells insights from nanomedicine into chloroquine efficacy against covid- identification of antiviral drug candidates against sars-cov- from fda-approved drugs (microbiology) fast gapped-read alignment with bowtie an exploratory randomized controlled study on the efficacy and safety of lopinavir/ritonavir or arbidol treating adult patients hospitalized with mild/moderate covid- (elacoi) the sequence alignment/map format and samtools hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro lipid rafts are involved in sars-cov entry into vero e cells hydroxychloroquine use against sars-cov- infection in non-human primates cutadapt removes adapter sequences from high-throughput sequencing reads ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to npc + endolysosomes is a rate-defining step inhibition of sars-cov- infections in engineered human tissues using clinical-grade soluble human ace . hydroxychloroquine-mediated inhibition of sars-cov- entry is attenuated by tmprss (microbiology) characterization of spike glycoprotein of sars-cov- on virus entry and its immune cross-reactivity with sars-cov repurposed antiviral drugs for covid- -interim who solidarity trial results stabilization of clathrin coated vesicles by amantadine, tromantadine and other hydrophobic amines baricitinib as potential treatment for -ncov acute respiratory disease discovery of sars-cov- antiviral drugs through large-scale compound repurposing hydroxychloroquine proves ineffective in hamsters and macaques infected with sars-cov- (pharmacology and toxicology) severe acute respiratory syndrome coronavirus replication is severely impaired by mg due to proteasome-independent inhibition of m-calpain inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry from sars to mers, thrusting coronaviruses into the spotlight covid- : combining antiviral and anti-inflammatory treatments a review of sars-cov- and the ongoing clinical trials mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro solidarity" clinical trial for covid- treatments clinical benefit of remdesivir in rhesus macaques infected with sars-cov- (microbiology) analysis of therapeutic targets for sars-cov- and discovery of potential drugs by computational methods development of novel entry inhibitors targeting emerging viruses we are grateful to patients at the hospital germans trias i pujol that donated their samples for research. for his excellent assistance and advice, we thank jordi puig from fundació lluita contra la sida. we are most grateful to lidia ruiz and the clinical sample management team of irsicaixa for their outstanding sample processing and management, and to m. pilar armengol and the translational genomics platform team at key: cord- -mwjh se authors: meng, fandan; suo, siqingaowa; zarlenga, dante s; cong, yingying; ma, xiaowei; zhao, qiong; ren, xiaofeng title: a phage-displayed peptide recognizing porcine aminopeptidase n is a potent small molecule inhibitor of pedv entry date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: mwjh se three phage-displayed peptides designated h, s and f that recognize porcine aminopeptidase n (papn), the cellular receptor of porcine transmissible gastroenteritis virus (tgev) were able to inhibit cell infection by tgev. these same peptides had no inhibitory effects on infection of vero cells by porcine epidemic diarrhea virus (pedv). however, when pedv, tgev and porcine pseudorabies virus were incubated with peptide h (hvtttfappppr), only infection of vero cells by pedv was inhibited. immunofluoresence assays indicated that inhibition of pedv infection by peptide h was independent of papn. western blots demonstrated that peptide h interacted with pedv spike protein and that pre-treatment of pedv with peptide h led to a higher inhibition than synchronous incubation with cells. these results indicate direct interaction with the virus is necessary to inhibit infectivity. temperature shift assays demonstrated that peptide h inhibited pre-attachment of the virus to the cells. coronaviruses belong to the family of coronaviridae and commonly cause respiratory or gastroenteric diseases (weiss and navas-martin, ; lai et al., ) . three groups of coronaviruses have been identified, based on differences in serology and genotyping (cavanagh, ; spaan et al., ) . these are enveloped viruses and consist of four major structural proteins: spike (s), membrane (m), nucleocapsid (n) and minor small envelop (e) protein (lai et al., ) . the host range and tissue tropism of coronaviruses depend on interactions between the viral s glycoprotein and receptors on susceptible cells (bosch et al., ; gallagher and buchmeier, ) . porcine transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhea virus (pedv) are swine-specific enteric coronaviruses that are antigenically distinguishable (lai et al., ; pensaert and yeo, ) . however, they replicate in the differentiated enterocytes of the small intestine resulting in similar clinical symptoms including lethal watery diarrhea and dehydration in piglets (pensaert and yeo, ; sanchez et al., ) . two decades ago, porcine aminopeptidase n (papn) was identified as a cellular receptor for tgev (delmas et al., ) . since that time, several limited reports have showed that addition of exogenous papn facilitates cell infection by pedv (li et al., ; oh et al., .) . recent evidence also indicates that increased papn receptor density on the surface of st cells contributes to cell infection by pedv (nam and lee, ) . the available data supports the hypothesis that blockage of papn is a good strategy for preventing cell infection by tgev or pedv. using the papn as a target protein, we identified three -mer peptides (designated as h, s or f) by phage display which bind to papn and competitively inhibit cell infection by tgev (ren et al., a) . the initial purpose of this study was to investigate the role of papn-binding peptides h, s and f on cell infection by pedv. interestingly, although there was no surface expression of papn on vero cells, peptide h decreased the infectivity of pedv in vitro. western blots indicated that peptide h (hvtttfappppr) interacted with the s protein of pedv. altering incubation temperatures further demonstrated that peptide h affected pre-attachment of pedv to cells. it is important to identify small molecules such as peptides that prevent infection by pedv, inasmuch as highly effective pedv vaccines which are currently not available. the peptide h identified herein may be one such candidate. concentration of compound that decreased the percentage of formazan produced in uninfected, peptide-treated cells to % of that produced in uninfected, peptide-free cells. the cc values were greater than μg/ml. all subsequent antiviral experiments were performed at peptide concentrations below the experimentally-determined cc value. in order to test the abilities of the three peptides to prevent attachment of pedv to cells, all combinations of peptide, virus and cell treatment were performed. cell post-treatment assays (fig. a) were performed to evaluate whether the three peptides were able to inhibit replication of pedv after infecting vero cells. plaque assays indicated that none of the three peptides inhibited pedv infection; however, rabbit anti-pedv decreased vial infectivity by more than % when the dilutions were reduced from : to : . when vero cells were pre-treated with peptide (cell pretreatment assay) prior to virus infection (fig. b) , little changes in virus titers were observed between the control and peptide treatment groups; some small effects were observed with the rabbit anti-pedv neutralizing antibodies. finally, in the virus pretreatment assays where pedv was incubated with peptides prior to cell infection (fig. c) , the results indicated that both peptides h and s inhibited pedv infectivity where ec values were approximately μg/ml and . μg/ml, respectively. the antiviral activity of peptide h was dose-dependent and at μg/ml it exhibited greater than % anti-pedv activity which is significantly higher than peptides s or f (p o . ); at . μg/ml, inhibition was greater than %. the selectivity indices si of peptides h and s were and , respectively. peptide f showed little inhibitory activity against pedv infection even at concentrations z μg/ml. inasmuch as papn may be involved in cell infection by pedv, the existence of papn on st, vero and mdck cells was analyzed by ifa. as shown in fig. , the endogenous papn expressed only on the surface of st cells, a porcine cell line. no expression was found on the surfaces of vero cells or mdck cells suggesting that the inhibitory activity of peptide h on pedv infection in vitro did not involve papn. the specificity of the inhibition of peptide h on pedv infection was assessed by comparing antiviral activities of peptide h on tgev and prv. further, peptide-induced cytotoxicity in st cells was also evaluated. results clearly show that peptide h had no demonstrable effects on tgev or prv even at very high peptide concentrations ( mm/ml) (fig. ) suggesting that a non-specific reactivity with virus envelopes is unlikely to be the cause for attenuating pedv infectivity. the effect of peptide h on the level of virus rna was quantified by real-time rt-pcr. the results demonstrated a dose-dependent decrease of viral rna synthesized in pedv-infected cells (fig. ) . at . μg/ml and . μg/ml, peptide h showed reduction of viral rna synthesis (po . ). however, at μg/ml, μg/ml and . μg/ml it significantly decreased viral rna synthesis (p o . ) when compared to the no peptide treatment group. the inhibitory activity of peptide h against pedv infection was confirmed by conventional rt-pcr. analysis of the pedv-rnas indicated that the density of the amplified sequences decreased with increasing concentrations of peptide h (data not shown). binding characteristics of the phage h (phage encoding peptide h) to pedv was analyzed by western blot. as shown in fig. , the phage h reacted with a protein with an approximate molecular mass of kda which is coincident with the molecular mass of the pedv s protein. antibody against the pedv s protein was used as a positive control. other controls including the m phage library and two phages bearing non-selected peptides did not react with the pedv s protein (fig. ) . these results indicate that the peptide h binds to the s protein of pedv. to further examine the mechanism of action of peptide h on cell infection by pedv, we investigated the effects of incubation temperature on cell infectivity. plaque assays showed that peptide infected with pedv at an pfu of  /ml. (c) peptides h, s or f were first incubated with pedv at c for h, and then the peptide treated viruses (pfu ¼  /ml) were used to infect vero cells at c. plaque assays were performed at the end of each experiment. serially-diluted polyclonal antibody against pedv and pbs were used as positive and negative controls, respectively. peptide concentrations , , , , and are μg/ml, μg/ml, . μg/ml, . μg/ml, and . μg/ml, respectively. anti-pedv antibody dilutions , , , , and are : , : ; : ; : ; and : , respectively. bars show the standard deviation from three independent assays. h exhibited significantly higher inhibitory effects (po . ) than peptide f or pbs on the pre-attachment of pedv to vero cells ( fig. a ) when virus was incubated with peptide h at c for h prior to incubation with the cells. when peptide h and pedv were co-incubated with vero cell at c for h before shifting to c (binding only), this allowed us to measure peptide h effects on early-and pre-attachment of the virus. results showed that pedv pre-treated with peptide h exhibited slightly higher inhibition (p . ) than when peptide h and pedv were co-incubated with cells absent a pre-incubation step (fig. b) suggesting that there is a direct effect of peptide h on pre-attachment. little to no inhibition of infection was observed once the virus became attached to the cell surface. furthermore, when the peptides and pedv were co-incubated with vero cells at c absent any preincubation step, no effective inhibition of cell infection was observed (fig. c ). to characterize the temperature effect on interactions between peptide h and pedv as well as cell infection, various concentrations of the peptides were incubated with pedv (pfu ¼  /ml) at c or c for h, then the peptide-treated viruses were used to infect cells at c. the results showed that the inhibition rate at c was significantly higher (p o . ) than that at c when the lower concentrations of peptide h were applied. the inhibition ratio reached . % at c but only . % at c at the lowest concentration ( . μg/ml). in contrast, high concentrations of peptide h gave rise to a similar inhibition of pedv infectivity (fig. d ). infection with tgev and pedv can cause high mortality in piglets and therefore enormous economic loss in the pig industry. the prevalence of pedv and tgev in asian countries such as china and korea has been documented (ren et al., b; li and ren, ) . at present, live vaccines against the both viruses are extensively used in china which in turn decreases the occurrence of diseases to some extent. however, small molecule inhibitors to tgev or pedv are alternative approaches to controlling swine viral diarrhea diseases. using combinatorial phage-display peptide libraries can be a powerful tool for selecting ligands that bind target proteins. phage display techniques have been used to generate diagnostic and therapeutic peptides for bacteria (bishop-hurley et al., carnazza et al., ) , fungi (bishop-hurley et al., ; fang et al., ) and viruses (ren et al., a ferrer and harrison, ; welch et al., ; wu et al., ; yang et al., ) . the papn is a member of a membrane-bound metalloprotease family and predominantly expressed on the surface of epithelial cells of the kidney, small intestine, and respiratory tract (nam and lee, ; kenny and maroux, ; lendeckel et al., ) . it is known that papn is a cellular receptor for tgev and that anti-papn antibody efficiently decreases cell infection by this virus (delmas et al., ; liu et al., ) . recently, three papn-binding peptides h, s, and f were identified using papn as an immobilized target for panning a -mer phage display peptide library. these peptides exhibited high affinity binding to papn and inhibited cell infection by tgev completely . as a member of group i coronaviruses, tgev and pedv have similar infection characterizations and as such it is difficult to differentiate these pathogens based only upon clinical symptoms. recent evidence indicates that pedv may also bind papn, a type ii glycoprotein, as a functional receptor (li et al., ; oh et al., ) . interestingly, tgev can be easily propagated in swine-originated cells such as st cells (delmas et al., ; hofmann and wyler, ) whereas pedv is adapted and cultivated in african green monkey kidney (vero) cells rather swine cells. given the stark similarities as well as differences between tgev and pedv, we were interested in evaluating the antiviral effects of the h, s and f peptides on cell infection by pedv. we first analyzed potential blockage of the papn-binding peptides on vero cell infection by pedv. plaque assays indicated no significant decrease in the infectivity of pdev even though prior studies showed that both anti-papn antibody and peptides h, s or f were capable of inhibiting tgev infection in vitro (ren et al., a; liu et al., ) if pre-incubated with tgev permissive cells. there were limited reports indicating that papn may serve as a functional receptor of pedv; however, studies herein clearly demonstrated that papn is only expressed on the surface of st cells and is not present on vero or mdck cells. as such, the inhibitory activities of peptide h were not due to binding papn. further, we showed that only pre-treatment with peptide h inhibited infection by pedv. the prv is a swine neurotropic herpesvirus with a dna genome that can be propagated in many cell lines including vero cells. therefore, we used prv as an additional control to further confirm and evaluate the inhibition specificity of the h peptide. peptide h did not prevent infection of pretreated tgev or prv suggesting its inhibitory activity was specific and not due to virucidal effects of amphipathic peptides. clearly, peptide h was able to interact with pedv; however, it was unclear as to the mechanism of its antiviral activity. as such, the binding characteristics between peptide h (using the phage bearing the h peptide) and pedv were further examined by western blot. results clearly showed that phage h reacted with a protein with a molecular mass of kda closely approximating the molecular mass of the pedv s protein. this supposition was corroborated using antibody against the pedv s protein. in contrast, control phages bearing other peptides did not show such reactivity. these results demonstrate that the peptide h abrogates infectivity in part by binding to the pedv s protein. this is consistent with the function of the coronavirus s protein that mediates cell infection. further, cell post-treatment assays evaluating the effects of each peptide on the replication of pedv in vitro relative amplification of the pedv x-n gene in the infected cells was normalized to that of beta-actin and calculated using the À ΔΔct method. peptide concentrations , , , , , and are μg/ml, . μg/ml, . μg/ml, . μg/ml, μg/ml, μg/ml, respectively; line is cell control group. displayed results are averages of three independent experiments. clearly demonstrated that the peptides do not interfere with the intracellular replication of pedv. our results showed that only peptide h and not peptides s or f exhibited very high, dose-dependent inhibitory activity against pedv where as little as μg/ml needed to achieve ec . this was confirmed by real-time rt-pcr which showed decreasing amounts of viral rna in pedv-infected cells. this corroborated the relationship between the antiviral activity of peptide h and either blockage of the viral attachment or entry into vero cells. the impact of peptide h on the entry of pedv was first investigated by performing the cell post-treatment and co-incubation assays. when pedv was incubated with cells prior to treatment with peptide h no significant effects on pedv infection were observed. similar results were seen when peptides, pedv and the cells were combined at the same time and co-incubated at c suggesting that peptide h did not affect pedv entry into the cell postadsorption. however, when pedv was pre-incubated with peptide h prior to incubation with vero cells, peptide h blocked the attachment of pedv as determined from plaque assays and rt-pcr analysis. it became clear that peptide h did not interact with vero cells directly. rather, it exhibits its inhibitory activity via the interplay between the peptide h and pedv. it is accepted that virus adsorption occurs at c and internalization does not happen until the temperature is raised to c (baldick et al., ) . our results clearly showed that the effects of peptide h occurred during the incubation step at c rather than the c internalization step again targeting a specific interaction between peptide h and pedv that affects binding to the cell surface. both pedv and tgev are group i coronaviruses (bridgen et al., ) and propagate in vero and st cells, respectively; prv is a dna virus that also propagates in vero cells. as such, we selected tgev and prv as control viruses to examine any specificity in the inhibitory activity of peptide h on cell infection by these viruses. as expected, the results showed no significant inhibitory activity of peptide h on either tgev or prv infection. further, peptide h was not cytotoxic to either st or vero cells. these results corroborate our hypothesis that peptide h functions in part, by interacting with the s protein of pedv and affecting the ability of the virus to bind to the cell surface. future studies will focus on identifying the specific site of interaction of peptide h and whether or not such a peptide can be used in vivo to abrogate or attenuate pedv infections. swine testis (st), monkey kidney cell lines (vero) and madin-darby canine kidney (mdck) cells were maintained in dulbecco's modified eagle medium (dmem) (invitrogen, us) supplemented with % fetal bovine serum, (fbs, gibco, us), units/ml of penicillin and units/ml of streptomycin. all cells were purchased from american type culture collection (atcc) and kept in our laboratory. pedv isolate hljby was propagated in vero cells in the presence of μg/ml trypsin (gibco, us) (ren et al., b) . tgev strain pur -mad was propagated in st cells (ren et al., ; yin et al., ) . porcine pseudorabies virus (prv) strain kaplan was propagated in vero cells (ren et al., c) . st or vero cells were seeded in -well plates at a density of  cells/well and cultured in dmem containing % fbs at c under % co for h followed by addition of serial dilutions ( . - μg/ml) of the tested peptides. the cells were allowed to grow for h at c and proliferation was analyzed by the -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt) method. briefly, the medium was removed and μl of mtt solution ( . mg/ml, invitrogen, us) was added and incubated at c for h. then μl of dimethyl sulfoxide (dmso) was added and incubated for min to solubilize the formazan crystals. cell survival rate was calculated as (od treatment)/(od control) (paeshuyse et al., ) . the % cytostatic concentration (cc ) was defined as the concentration inhibiting the proliferation of (b) pedv (pfu ¼  /ml), peptides h or f, and vero cells were co-incubated at c for h, washed, then shifted to c. (c) pedv (pfu ¼  /ml), peptides, and vero cells were co-incubated at c for h then washed prior to assaying. (d) pedv was treated with various concentrations of peptides h or f at c or c for h then used to infect vero cells (pfu ¼  /ml) at c. peptide concentrations , , , , and are μg/ml, μg/ml, . μg/ml, . μg/ml, and . μg/ml, respectively. anti-pedv antibody dilutions , , , , and are : , : , : , : and : , respectively. plaque assays were further performed at h post-infection. bars show the standard deviation from three independent assays. exponentially growing cells by %. non-cytotoxic concentrations of peptides (r cc ) were used for antiviral assays. three different treatment approaches were used to analyze the antiviral action of the selected peptides h (hvtttfappppr), s (svvpskatwgfa) and f (fkpssppsitlw) as previously defined (kwon et al., ) . in the first method, i.e., cell post-treatment assay, vero cells were grown in -well plates at a density of  cells/well for h. pedv at a pfu (plaque-forming unit) of  /ml was inoculated onto confluent cells for h followed by removal of the medium and incubation of the infected cells with various noncytotoxic concentrations (r cc ) of peptides for h at c. the cells were then overlaid with % methylcellulose in dmem and incubated for h at c followed by crystal violet staining and plaque assays as previously described with modifications (ren et al., a (ren et al., , d . briefly, after the medium was removed, the cells were fixed with % formaldehyde in pbs for h at room temperature followed by staining with % crystal violet solution for min. the staining solution was removed, the cells were washed with pbs and the plaques were counted. decreases in virus infectivity were calculated from the plaque assay as follows:  [ À (treatment wells/control wells)]. average ec values (concentration inducing % inhibition of virus replication) were calculated and the effectiveness of each peptide were expressed using the selectivity index (si) (si¼ cc /ec ) (paeshuyse et al., ; müller et al., ) . in the second method, i.e., cell pre-treatment assay, vero cells were first grown in -well plates at a density of  cells/well for h then treated with non-cytotoxic concentrations of peptide for h prior to incubation with virus. the peptides were removed and the cells were washed twice with pbs. pedv at a pfu of  /ml was inoculated onto the pre-treated vero cells for h. after the virus was removed, the cells were overlaid with % methylcellulose in dmem and incubated for h at c followed by plaque assays. in the third experiment i.e., virus pre-treatment, various concentrations of the peptides were mixed with pedv (pfu ¼  /ml) at c for h prior to incubation with cells. vero cells were grown in -well plates at  cells/well for h then the peptide/virus mixture was added to the cultured cells for h. after the mixture was removed and the cells washed with pbs, the cells were overlaid with % methylcellulose in dmem and cultured for an additional h at c followed by plaque assays as described above. in parallel, pedv-neutralizing, rabbit antiserum serially-diluted : and pbs were used as positive and negative controls, respectively, for the above-mentioned experiments. each concentration of the peptide and antibody was assayed in triplicate. immunofluorescence assays to identify papn on cell lines from different species st, vero and mdck cells were seed into the -well plates and incubated at c for h. indirect immunofluorescence assays (ifa) were performed (ren et al., d; baldick et al., ) with modification. the cells were washed twice with pre-chilled pbs then fixed with % paraformaldehyde in pbs for min at room temperature. following two washes with pbs, they were quenched with . m glycine for min then blocked with % bsa (sigma, us) in pbs for min. samples were incubated for h with anti-papn polyclonal antibody ( : in pbs) (liu et al., ) , washed three times with pbs, and incubated with fluorescein isothiocyanate (fitc) conjugated goat anti-rabbit igg ( : in pbs, zhongshan, china) for h in the dark. the samples were washed three times with pbs and the fluorescence signals and phase contrast images were detected by fluorescence microscopy (leica, wetzlar, germany). various concentrations of peptide h were incubated with the tgev, pedv and prv (pfu¼  /ml) at c for h then added to confluent vero or st cell monolayers for h. the mixtures were removed and the cultured cells washed twice with pbs then incubated with % methylcellulose in dmem for h at c. the cells were then stained with crystal violet staining and plaque assays were performed as described above. the effect of peptide h on pedv infection of vero cells was confirmed by semi-quantitative real-time reverse transcription (rt-pcr) (ren et al., d) . vero cells in -well plates were infected with pedv (pfu ¼  /ml) pretreated with different concentrations of peptide h at c for h. the culture was replaced with dmem at c for h then washed times with pbs. the virus-containing cultures were frozen and thawed three times followed by addition of an equal volume of % polyethylene glycol (pge) at room temperature and incubation for min. the samples were centrifuged at , rpm for min and the pellets were re-suspended in rnase-free water. total rna was extracted with a commercial kit (fastgene, china) according to the manufacturer's instructions. reverse transcription was performed in a total of μl consisting of μl total rna ( . μg), μl oligo dt, μl dntp ( mm), . μl rnase inhibitor, . μl sterile water, μl mlv, and μl  rt-pcr buffer. the mixture was incubated at c for min, c for min and c for min. real-time pcr was performed using abi prism real-time pcr machine (applied biosystems, usa). the information regarding primers and rt-pcr products is provided in table . the real-time pcr mixture included . μl ( . μg) of cdna template, μl of sybr taq polymerase, . μl of rox pge , . μl ( pmol) of each primer, and . μl of sterile water. the reactions were incubated at c for s followed by cycles of c for s and c for s. we examined virus rna levels using primers that specifically amplify a bp fragment encompassing the ' end of a small, non-structural gene (x) and the ' end of the pedv-n gene ( table ). the expression of pedv x-n in pedv-infected vero cells was normalized to that of beta-actin and taken as % compared to expression of the peptide h treatment group. data analysis is based on the measurement of the cycle threshold (ct). the difference in Δct (ct sample fragment mean ct value-beta-actin fragment mean ct value) was used as a measure of the relative fragment with the À ΔΔct method in correlation to the amplification size of pedv x-n fragment. for each experimental condition, real-time pcr was conducted in quadruplicate and the resulting average ct values for the pedv x-n fragment was used to quantify viral load. the experiment was performed in triplicate. information on primers and real-time rt-pcr products. sense ' cactggttgggctttctatgtc pedvx-n antisense 'tgttagtgggtacagcgttgtt sense ' ggctcagagcaagagaggtatcc β-actin antisense ' ggtctcaaacatgatctgagtcatct western blot analysis of peptide h binding to pedv the pedv (  pfu/ml) was harvested from vero cells and clarified by centrifugation at g for min followed by ultracentrifugation at , g for . h to collect the virus. the pellets were suspended in pbs, subjected to % sds-page then blotted to a nitrocellulose (nc) membrane. the nc membranes were blocked overnight at c with % non-fat dry milk in pbs. after three washes with pbs, membranes were sliced and incubated with phage h (  pfu), anti-pedv s polyclonal antibody ( : in pbs), m phage library (  pfu), and control phage bearing either the peptides svsvgmkpsprp or mscndtlcllpn. the membranes were then washed with pbs and successively incubated with anti-m polyclonal antibody (abcam, : in pbs) and horseradish peroxidase hrp-conjugated goat anti-rabbit igg ( : in pbs) at room temperature for h. protein bands were visualized using , '-diaminodbenzidine (dab, the thermo scientific) to examine the effect of temperature on the binding of virus to the cells, four experiments were performed. first, various concentrations of peptide h and control peptide f were incubated with pedv (pfu¼  /ml) at c for h then added to confluent vero cells seeded in -well plates at c for h followed by infection at c for h. second, the peptides, pedv and vero cells were co-incubated at c for h, after which the incubation temperature was raised to c for h. third, the peptides, pedv and vero cells were co-incubated at c for h then assayed without prior incubation at c. finally, peptides were pre-incubated with pedv at c or c for h followed by cell infection at c for h. all experiments were terminated by extensive washing of the cells and plaque assays to quantify the infection. statistical analysis of the data was performed using spss . software; p o . and p o . were defined as statistically significant and highly statistically significant, respectively. a novel small molecule inhibitor of hepatitis c virus entry phage-displayed peptides as developmental agonists for phytophthora capsici zoospores phage-displayed peptides selected for binding to campylobacter jejuni are antimicrobial peptides selected for binding to a virulent strain of haemophilus influenzae by phage display are bactericidal the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex sequence determination of the nucleocapsid protein gene of the porcine epidemic diarrhoea virus confirms that this virus is a coronavirus related to human coronavirus e and porcine transmissible gastroenteritis virus specific and selective probes for pseudomonas aeruginosa from phage-displayed random peptide libraries nidovirales, a new order comprising coronaviridae and arteriviridae aminopeptidase n is a major receptor for the enteropathogenic coronavirus tgev combinatorially selected defense peptides protect plant roots from pathogen infection peptide ligands to human immunodeficiency virus type gp identified from phage display libraries coronavirus spike proteins in viral entry and pathogenesis propagation of the virus of porcine epidemic diarrhea in cell culture topology of microvillar membrance hydrolases of kidney and intestine in vitro inhibitory activity of alpinia katsumadai extracts against influenza virus infection and hemagglutination coronaviridae review: the role of membrane peptidases in immune functions porcine aminopeptidase n is a functional receptor for the pedv coronavirus reverse transcription loop-mediated isothermal amplification for rapid detection of transmissible gastroenteritis virus expression and functional analysis of porcine aminopeptidase n produced in prokaryotic expression system evaluation of antiviral activity of south american plant extracts against herpes simplex virus type and rabies virus contribution of the porcine aminopeptidase n (cd ) receptor density to porcine epidemic diarrhea virus infection identification of a putative cellular receptor kda polypeptide for porcine epidemic diarrhea virus in porcine enterocytes a novel, highly selective inhibitor of pestivirus replication that targets the viral rna-dependent rna polymerase porcine epidemic diarrhea importance of cholesterol for infection of cells by transmissible gastroenteritis virus phage displayed peptides recognizing porcine aminopeptidase n inhibit transmissible gastroenteritis coronavirus infection in vitro action mechanisms of lithium chloride on cell infection by transmissible gastroenteritis coronavirus development of a porcine epidemic diarrhea virus m protein-based elisa for virus detection phages harboring specific peptides that recognize the n protein of the porcine reproductive and respiratory syndrome virus distinguish the virus from other viruses cholesterol dependence of pseudorabies herpesvirus entry genetic evolution and tropism of transmissible gastroenteritis coronavirus virus taxonomy, eighth report of the international committee on taxonomy of viruses coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol potent dpeptide inhibitors of hiv- entry phage displayed peptides to avian h n virus distinguished the virus from other viruses potent suppression of viral infectivity by the peptides that inhibit multimerization of human immunodeficiency virus type i (hiv- ) vif proteins cholesterol is important for a post-adsorption step in the entry process of transmissible gastroenteritis virus this work was supported by national natural science foundation of china ( and ), sponsored by chang jiang scholar candidates program for provincial universities in heilongjiang ( cjhb ), the program for new century excellent talents in university of ministry of education of p.r. china (ncet- - ). key: cord- -hjx d rq authors: márquez-jurado, silvia; nogales, aitor; Ávila-pérez, ginés; iborra, francisco j.; martínez-sobrido, luis; almazán, fernando title: an alanine-to-valine substitution in the residue of zika virus ns a protein affects viral rna synthesis and attenuates the virus in vivo date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: hjx d rq the recent outbreaks of zika virus (zikv), its association with guillain–barré syndrome and fetal abnormalities, and the lack of approved vaccines and antivirals, highlight the importance of developing countermeasures to combat zikv disease. in this respect, infectious clones constitute excellent tools to accomplish these goals. however, flavivirus infectious clones are often difficult to work with due to the toxicity of some flavivirus sequences in bacteria. to bypass this problem, several alternative approaches have been applied for the generation of zikv clones including, among others, in vitro ligation, insertions of introns and using infectious subgenomic amplicons. here, we report a simple and novel dna-launched approach based on the use of a bacterial artificial chromosome (bac) to generate a cdna clone of rio grande do norte natal zikv strain. the sequence was identified from the brain tissue of an aborted fetus with microcephaly. the bac clone was fully stable in bacteria and the infectious virus was efficiently recovered in vero cells through direct delivery of the cdna clone. the rescued virus yielded high titers in vero cells and was pathogenic in a validated mouse model (a mice) of zikv infection. furthermore, using this infectious clone we have generated a mutant zikv containing a single amino acid substitution (a v) in the ns a protein that presented reduced viral rna synthesis in cell cultures, was highly attenuated in vivo and induced fully protection against a lethal challenge with zikv wild-type. this bac approach provides a stable and reliable reverse genetic system for zikv that will help to identify viral determinants of virulence and facilitate the development of vaccine and therapeutic strategies. zika virus (zikv) is a recently emerged mosquito-borne member of the family flaviviridae, which was declared by the word health organization (who) as a global public health emergency on february [ , ] . like other flaviviruses, the viral particle is constituted by an inner nucleocapsid composed of the capsid (c) protein associated with the viral genomic rna (grna), surrounded by a lipid bilayer that contains the structural membrane (m) and envelope (e) proteins, which are arranged nucleus from the cmv promoter with a second amplification step in the cytoplasm driven by the viral polymerase. the recombinant virus rescued from the bac clone was fully infectious in vitro and in vivo. the zikv-rgn infectious clone was further used to evaluate the effect of a single amino acid change (alanine to valine) at residue of the ns a protein on viral rna synthesis and pathogenesis in vivo. we found that this unique single amino acid substitution impairs viral rna synthesis in cell culture and results in viral attenuation in a mice. remarkably, a single dose of the mutant virus was sufficient to induce protection against challenge with the parental wild-type (wt) zikv. these results demonstrate the reliability and potential of our bac approach to study zikv biology and to facilitate the development of vaccine and antiviral strategies. vero (a kidney epithelial cell line from an african green monkey) and a (an human adenocarcinomic alveolar epithelial cell line) cells were purchased from the american type culture collection (atcc, ccl- ) and were grown and maintained at • c and % co in growth medium, consisting in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs) (hyclone, thermofisher scientific, madrid, spain), mm l-glutamine (sigma-aldrich, madrid, spain), % nonessential amino acids (sigma-aldrich), u/ml penicillin (sigma-aldrich) and µg/ml streptomycin (sigma-aldrich). the recombinant zikv-rgn wt (rzikv-rgn) or ns a mutant (rzikv-rgn-mns a) viruses were propagated in vero cells with virus growth medium (dmem supplemented with % fbs, mm l-glutamine, % nonessential amino acids, u/ml penicillin and µg/ml streptomycin) at • c and % co . for virus stocks preparation, to % confluent monolayers of vero cells were infected with a multiplicity of infection (moi) of . plaque forming units (pfu) per cell in virus growth medium and incubated at • c under % co . after - days of infection, the tissue culture supernatants were collected, clarified by centrifugation at × g for min, and stored in small aliquots at − • c. the bac plasmid pbelobac [ ] , kindly provided by h. shizuya (california institute of technology, pasadena, ca, usa), was used to assemble the zikv-rgn infectious cdna clone. this plasmid is a synthetic low-copy-number plasmid (one copy per cell) based on the escherichia coli (e. coli) f-factor [ ] that minimize the toxicity problems in the bacteria of exogenous sequences. e. coli dh b cells (invitrogen, thermofisher scientific) were used to amplify the bac plasmids. electrocompetent dh b cells (invitrogen, thermofisher scientific) were transformed by electroporation using a micropulser unit (bio-rad, madrid, spain), according to the manufacturer's instructions. bac-based plasmids were isolated and purified using the large-construct kit (qiagen, hilden, germany), following the manufacturer's specifications. we have assembled a zikv infectious cdna clone in the bac plasmid pbelobac , based on the data of the full-length sequence of the zikv clinical strain rgn [ ] deposited in the genbank (accession number ku ). this strain was selected because the full-length sequence was obtained directly from the virus-infected brain tissue of an aborted fetus with microcephaly in brazil in and therefore represents a good candidate to study zikv pathogenesis. the first step for the assembly of the full-length cdna clone was the selection of the restriction sites pmli, afei, and bstbi (genomic positions , and , respectively), which are unique in the viral genome ( figure a ). after that, four overlapping dna fragments covering the entire viral genome (zikv to zikv ) and flanked by the appropriate restriction sites, were generated by chemical synthesis (bio basic, inc., toronto, canada) ( figure b ). zikv fragment contained the cmv promoter precisely fused to the first nucleotides of the viral genome flanked at the '-end by apali and asci (absent in the viral genome) sites and at the '-end by a multiple-cloning site containing the selected restriction sites (pmli, afei and bstbi) followed by mlui (absent in the viral genome) and bamhi. fragments zikv (flanked by pmli and afei) and zikv (flanked by afei and bstbi) covered the genomic regions - and - , respectively. zikv fragment contained the restriction site bstbi, the last nucleotides of the viral genome, the hepatitis delta virus (hdv) ribozyme, the bovine growth hormone (bgh) termination and polyadenylation sequences, and the mlui restriction site. the infectious clone was assembled into pbelobac by sequential cloning of these four overlapping dna fragments. briefly, fragment zikv was digested with apali and bamhi and cloned into pbelobac −afei (a pbelobac without the afei restriction site) digested with the same enzymes, to generate the intermediate plasmid pbac-zikv . then, this plasmid was used as the backbone for the sequential cloning of the remaining overlapping dna fragments (zikv to zikv ) into the multicloning site of the intermediate plasmid (contains the restriction sites selected, pmli, afei, bstbi and mlui) to generate the full-length cdna clone pbac-zikv-rgn ( figure b) . the genetic integrity of the cdna clone was verified throughout the assembly process by extensive restriction analysis and sequencing. in all cases, the bacterial strain dh b (invitrogen, thermofisher scientific) was used as the e. coli host for all the cloning steps and the propagation of the bac cdna clone. to recover the infectious virus, vero cells on -well plates were grown to % confluence in growth medium without antibiotics, and transfected with µg of the bac cdna clone using µl of lipofectamine (invitrogen, thermofisher scientific), following the manufacturer's specifications. after h of incubation at • c, the transfection medium was replaced with fresh growth medium and the cells incubated at • c. aliquots of the culture supernatants were collected at h intervals for virus titer determination by plaque assay on vero cells. after five to seven days of transfection, when the cytopathic effect (cpe) was clear, cell culture supernatants were harvested and the recovered virus was cloned by three rounds of plaque purification. to determine the complete genome sequence of the rescued viruses, virions from supernatant of infected vero cells (moi of . pfu/cell) were purified through a % (w/v) sucrose cushion. viral rna was isolate from the purified virus with the qiaamp viral rna minikit (qiagen) following the manufacturer's instructions and deep-sequenced at the university of rochester genomics research center using illumina miseq (illumina, san diego, ca, usa). briefly, . µg of total viral rna was fragmented by controlled sonication and a dna library was generated using the nebnext mrna library prep master mix set for illumina (new england biolabs, ipswich, ma, usa), according to the manufacturer's instructions. after analyzing the library for size and quality (bio-analyzer; agilent technologies, inc., santa clara, ca, usa), deep-sequencing was performed using miseq (illumina) and the raw sequencing reads analyzed using swarm custom software. the genomic 'and '-terminal sequences were determined by the rapid amplification of cdna ends (race) using the '/ ' race second generation kit (roche, basilea, switzerland) with a polya-tail added to the cdna prior to the ' race reaction using polya polymerase (new england biolabs), following the manufacturer's instructions. to analyze the genetic stability of the recombinant zikv harboring the point mutation a v in the coding region of the ns a protein (rzikv-rgn-mns a), total rna was purified from vero cells infected with viruses from passage (p ) to passage (p ) using the rneasy minikit (qiagen), according to the manufacturer's specifications. purified rna ( ng) was reverse transcribed (rt) with random hexamer primers using the high-capacity cdna transcription kit (life technologies, thermofisher scientific), and the cdna was amplified by pcr with the forward primer zikv- vs viruses , , of ( '-gaggaatggtgctgcagg- '), spanning nucleotides to of the viral genome, and the reverse primer zikv- rs ( '-gcttgacatctccccag- ') , complementary to nucleotides to of the viral genome. finally, the amplicons generated covering the region encoding ns a and ns b proteins (genomic region - ) were sequenced by sanger sequencing (macrogen europe, amsterdam, netherlands) using specific oligonucleotides. vero cells seeded into -well plates at - % of confluence were infected with µl of serial -fold dilutions of the virus in virus growth medium without fbs for h at • c. after viral absorption, the viral inoculum was removed and the cells overlaid with ml of virus growth medium containing % deae-dextran (sigma-aldrich) and . % agar noble (difco, thermofisher scientific). after - days of incubation at • c under % co , the cells were fixed with % formaldehyde for h at room temperature, the overlaid removed, and the viral plaques visualized by staining with . % crystal violet in % methanol or by immunostaining with µg/ml of the pan-flavivirus e protein monoclonal antibody (mab) g (bei resources; nr- ) using the vectastain abc kit (vector laboratories inc., burlingame, ca, usa). visible plaques were counted and virus titers were calculated as pfu/ml. vero and a cells seeded into -well plates at % of confluence were infected with the indicated viruses diluted in virus growth medium without fbs at the specified mois. after h of absorption at • c in % co , the virus inoculum was removed, the cell monolayers washed twice with pbs, and . ml of fresh virus growth medium was added to each well. cells were incubated at • c under % co and at selected time points, aliquots of tissue culture supernatants were collected and virus titers determined by plaque assay in vero cells as described above. viral rna synthesis was evaluated by quantitative rt-pcr (rt-qpcr). total intracellular rna from uninfected or infected vero cells was purified using the rneasy minikit (qiagen) and total cdna was synthetized from ng of purified rna using random hexamer primers and the high-capacity cdna transcription kit (life technologies, thermofisher scientific), following the manufacturer's specifications. using this cdna, the level of viral rna was further quantified by qpcr using a custom taqman assay specific for zikv-rgn rna. this taqman assay is constituted by the forward primer '-gaagagcatccagccagagaa- ' (spanning nucleotides to of the viral genome), the reverse primer '-ctgggagccatgaactgaca- ' (complementary to nucleotides to of the viral genome), and the probe '-fam-tggagtaccggataatg- iabkfq- ' (covering nucleotides to of the viral genome). to normalize for differences in rna sampling, the expression of the histone h b (reference housekeeping gene) was analyzed using a specific taqman gene expression assay (rh _s ; life technologies, thermofisher scientific). data were acquired with a real-time pcr system (life technologies, thermofisher scientific) and analyzed with abi prism software v . . . the relative quantifications were performed using the cycle threshold ( −∆∆ct ) method [ ] . all experiments and data analysis were miqe (minimum information for publication of quantitative real-time pcr experiments) compliant [ ] . the expression of zikv e protein was analyzed by indirect immunofluorescence assay (ifa). vero cells grown on coverslips in -well plates at - % of confluence were infected with the rescued rzikvs at the indicated mois. at selected time points post-infection, cells were fixed with % paraformaldehyde in mm hepes ph . during min at room temperature and then permeabilized with . % triton x- in pbs for min. after that, cells were treated for h at room temperature with blocking solution ( % fbs in pbs) and incubated with µg/ml of the pan-flavivirus e protein mab g (bei resources; nr- ) in blocking solution for h at room temperature. after three washed with pbs, cells were incubated at room temperature for h with donkey anti-mouse antibody conjugated to alexa fluor (invitrogen, thermofisher scientific) diluted : in blocking solution, extensively washed with pbs, and incubated for min with dapi ( ', '-diamidino- -phenylindole) (sigma-aldrich) diluted : in pbs for nuclear staining. finally, coverslips were mounted in prolong gold antifade reagent (invitrogen, thermofisher scientific) and analyzed on a leica sp confocal microscope. immunofluorescence acquired images were processed and analyzed with imagej . b software [ ] . for the evaluation of the virus-specific antibodies levels present in the sera of vaccinated mice, enzyme-linked immunosorbent assays (elisas) were performed as previously described [ ] . briefly, -well plates were coated with cell lysates from mock-or zikv-infected vero cells and incubated overnight at • c. the coated wells were washed with pbs, blocked with % bsa in pbs, and then incubated with two-fold dilutions (starting dilution of : ) of mice sera for h at • c. after that, plates were washed with water and incubated with hrp-conjugated goat anti-mouse igg ( : ; southern biotech, birmingham, al, usa) for h at • c. reactions were developed with tetramethylbenzidine (tmb) substrate (biolegend, san diego, ca, usa) for min at room temperature, quenched with n h so , and read at nm in a vmax kinetic microplate reader (molecular devices, san jose, ca, usa). the in vivo studies were performed in type-i interferon (ifn) receptor deficient (ifnr-/-) a mice (the jackson laboratory, bar harbor, me, usa) maintained in the animal care facility at the university of rochester under specific pathogen-free conditions. in this animal model, subcutaneous (s.c.) or intraperitoneal (i.p.) infection with zikv induces neurological disease and the animals succumb to viral infection, with high viral load in blood, brain, spin cord, and testes, consistent with manifestations of zikv infection in humans. although deficient in innate ifn responses, a mice retain their adaptive immunity and have been successfully used as a suitable model for testing antivirals and vaccines [ ] [ ] [ ] . to evaluate virus pathogenicity, female -to- -week-old a mice (n = ) were first anesthetized i.p. with a mixture of ketamine ( µg per gram of body weight) and xylazine ( µg per gram of body weight), and then mock-infected (pbs) or infected s.c. in the footpad with the indicated doses of rzikv-rgn or rzikv-rgn-mns a diluted in pbs in a final volume of µl. after viral infection, animals were monitored daily for morbidity (body weight loss and disease signs, including hunching, ruffling and hind limb paralysis) and mortality (survival) over days. mice showing more than % of body weight loss or severe paralysis were considered to have reached the experimental endpoint and were humanely euthanized. to correlate development of clinical symptoms and death with virus replication, -to- -week-old mice (n = ) were infected as described above and the viral titers in serum were determined at days (n = ) and (n = ) by plaque assay and immunostaining using the pan-flavivirus e protein mab g as indicated before. to evaluate the protection efficacy of the rzikv-rgn-mns a, female -to- -week-old a mice (n = ) were first anesthetized i.p. as indicated above, and then mock-immunized (pbs) or immunized s.c. in the footpad with pfu of rzikv-rgn-mns a diluted in pbs in a final volume of µl. at days post-immunization, mouse sera were collected by submandibular bleeding and the presence of total antibodies against zikv-rgn was evaluated by elisa. twenty-four hours after bleeding, mice were challenged s.c. in the footpad with pfu of rzikv-rgn and their morbidity and mortality monitored over days as previously described. to determine viral replication, challenged -to- -week-old a mice (n = ) were bleeding at days (n = ) and (n = ) post-challenge and viruses , , of zikv viremia was determined by plaque assay and immunostaining using the pan-flavivirus e protein mab g as previously described. for quantitative analyses, a two-tailed, unpaired student t test was used to analyze differences in mean values between groups. all results were expressed as mean ± standard deviations of the means. p values of < . were considered significant. for mice experiments, the meier log-rank test was used to compare survival data and the reed and muench method to determine the mouse lethal dose (mld ). graphpad prism v . software was used for all statistical analysis. all animal protocols were approved by the university of rochester committee of animal resources (protocol number: ucar- - / ; approval date: / / ) and complied with the recommendations in the guide for the care and use of laboratory animals of the national research council [ ] . to overcome the toxicity problems associated to several flavivirus sequences during its propagation in bacteria, we used the bac plasmid pbelobac (a single-copy plasmid derived from the e. coli f-factor) [ ] to assemble a zikv infectious cdna clone, based on the genome sequence of the rgn strain of zikv (genbank accession number ku ) [ ] (figure ). this zikv-rgn strain was selected because it has no laboratory passage history and the full-length genome sequence was obtained from a zikv-infected fetus with microcephaly in [ ] , constituting a good candidate to further study zikv pathogenesis. after appropriate selection of unique restriction sites in the zikv-rgn genome ( figure a ), four overlapping dna fragments (zikv to zikv ), spanning the full-length viral genome and flanked for the selected restriction sites, were chemically synthesized, and sequentially cloned into pbelobac to generate the infectious cdna clone pbac-zikv-rgn ( figure b ). fragment zikv contained the cmv immediate-early promoter to allow the expression of the viral rna in the nucleus by the cellular rna polymerase ii [ ] and fragment zikv was flanked at the '-end by the hdv ribozyme followed by the bgh termination and polyadenylation sequences to produce synthetic rnas bearing authentic '-ends of the viral genome. this dna-lunched system ensures capping of the viral rna and allows the recovery of infectious virus from the transfected cdna clone without the need of an in vitro transcription step. once assembled, the full-length sequence of the zikv-rgn bac clone was determined and no changes were detected to that reported for the zikv-rgn strain (genbank accession number ku ). finally, to confirm the stability of this synthetic infectious cdna clone in bacteria, the bac clone was passaged in e. coli dh b cells for more than two hundred generations and the genetic integrity of the passaged infectious clone analyzed by restriction endonuclease analysis and sequencing. no differences were detected, demonstrating that the zikv-rgn bac clone was fully stable in bacteria and that the bac approach is a reliable and simple method to generate zikv infectious cdna clones. to recover the infectious virus ( figure ), vero cells were transiently transfected with the bac cdna clone using lipofectamine and virus production analyzed during seven days. in contrast to mock-transfected cells, increasing amounts of infectious virus were detected in the tissue culture supernatant of cells transfected with the infectious clone, with peak titers around pfu/ml on day five ( figure a ). to further confirm the identity of the rescued virus, vero cells were infected with an moi of . pfu/cell of the rescue virus and monitored for cpe induction and viral e protein expression by ifa using the pan-flavivirus e protein mab g ( figure b ). the rescued virus induced a clear cpe, characterized by the presence of rounded and birefringent cells, and high levels of e protein expression were detected in the perinuclear region of infected cells. these results demonstrated that the zikv-rgn infectious bac cdna clone produces high titers of rzikv-rgn directly after transfection of susceptible vero cells. to recover the infectious virus ( figure ), vero cells were transiently transfected with the bac cdna clone using lipofectamine and virus production analyzed during seven days. in contrast to mock-transfected cells, increasing amounts of infectious virus were detected in the tissue culture supernatant of cells transfected with the infectious clone, with peak titers around pfu/ml on day five (figure a ). to further confirm the identity of the rescued virus, vero cells were infected with an moi of . pfu/cell of the rescue virus and monitored for cpe induction and viral e protein expression by ifa using the pan-flavivirus e protein mab g ( figure b ). the rescued virus induced a clear cpe, characterized by the presence of rounded and birefringent cells, and high levels of e protein expression were detected in the perinuclear region of infected cells. these results demonstrated that the zikv-rgn infectious bac cdna clone produces high titers of rzikv-rgn directly after transfection of susceptible vero cells. once the identity of the rescued virus was confirmed, it was cloned by three round of plaque purification, and its phenotypic and genotypic properties were determined. analysis of the growth kinetics revealed that the rzikv-rgn replicated efficiently in both vero and a cells, reaching peak titers of approximately and pfu/ml at hpi, respectively ( figure a ). in addition, the rescued virus generated homogeneous plaques of about mm in size after four days of infection in vero cells ( figure b ). finally, the genetic identity of the virus was analyzed by deep-sequencing of two independent clones. full-genome sequencing of both viral clones revealed that both clones presented the same sequence that the cdna clone. overall, these results demonstrate the feasibility of generating infectious rzikvs using a bac-based approach. once the identity of the rescued virus was confirmed, it was cloned by three round of plaque purification, and its phenotypic and genotypic properties were determined. analysis of the growth kinetics revealed that the rzikv-rgn replicated efficiently in both vero and a cells, reaching peak titers of approximately and pfu/ml at hpi, respectively ( figure a ). in addition, the rescued virus generated homogeneous plaques of about mm in size after four days of infection in vero cells ( figure b ). finally, the genetic identity of the virus was analyzed by deep-sequencing of two independent clones. full-genome sequencing of both viral clones revealed that both clones presented the same sequence that the cdna clone. overall, these results demonstrate the feasibility of generating infectious rzikvs using a bac-based approach. once the identity of the rescued virus was confirmed, it was cloned by three round of plaque purification, and its phenotypic and genotypic properties were determined. analysis of the growth kinetics revealed that the rzikv-rgn replicated efficiently in both vero and a cells, reaching peak titers of approximately and pfu/ml at hpi, respectively ( figure a ). in addition, the rescued virus generated homogeneous plaques of about mm in size after four days of infection in vero cells ( figure b ). finally, the genetic identity of the virus was analyzed by deep-sequencing of two independent clones. full-genome sequencing of both viral clones revealed that both clones presented the same sequence that the cdna clone. overall, these results demonstrate the feasibility of generating infectious rzikvs using a bac-based approach. to determine whether the rescued rzikv-rgn was pathogenic in vivo (figure ) , groups of five female -to- -week-old a mice (ifnr-/-) were inoculated s.c. in the footpad with pbs (as negative control) or with different doses of rzikv-rgn ( , and pfu per animal) and the morbidity (body weight loss and disease signs) and survival were monitored daily over days ( figure a ). as expected, weight loss and survival correlated with the inoculated dose. mice infected with pfu did not show disease symptoms, only a slight reduction in body weight was detected on days to , and all of them survived. in the case of mice infected with pfu, they presented some symptoms of disease (hunching and reduced mobility) and weight loss from days seven to nine (with a maximum of % on day nine), but all of them recovered the initial body weight and survived. in contrast, animals infected with pfu showed hind limb paralysis, rapidly lost weight, and all of them succumbed to viral infection between days seven and eight post-infection ( figure a ). using the reed & muench method we determined that the mld of rzikv-rgn was approximately × pfu. to further analyze whether the virulence observed correlated with viral replication, viral titers in mouse sera were analyzed at days two and four post-infection ( figure b ). as expected, the viremia in the infected animals was dose dependent, reaching the highest titers at day two after inoculation. at day four post-inoculation a significant reduction of the viral titers was observed. in the case of animals infected with or pfu, viremia was not detected or only detected in one of the three infected mice, respectively ( figure b ). overall, these results indicated that the rzikv-rgn recovered from the infectious clone is virulent in mice but only at high ( pfu) dose. to determine whether the rescued rzikv-rgn was pathogenic in vivo (figure ) , groups of five female -to- -week-old a mice (ifnr-/-) were inoculated s.c. in the footpad with pbs (as negative control) or with different doses of rzikv-rgn ( , and pfu per animal) and the morbidity (body weight loss and disease signs) and survival were monitored daily over days ( figure a ). as expected, weight loss and survival correlated with the inoculated dose. mice infected with pfu did not show disease symptoms, only a slight reduction in body weight was detected on days to , and all of them survived. in the case of mice infected with pfu, they presented some symptoms of disease (hunching and reduced mobility) and weight loss from days seven to nine (with a maximum of % on day nine), but all of them recovered the initial body weight and survived. in contrast, animals infected with pfu showed hind limb paralysis, rapidly lost weight, and all of them succumbed to viral infection between days seven and eight post-infection ( figure a ). using the reed & muench method we determined that the mld of rzikv-rgn was approximately × pfu. to further analyze whether the virulence observed correlated with viral replication, viral titers in mouse sera were analyzed at days two and four post-infection ( figure b ). as expected, the viremia in the infected animals was dose dependent, reaching the highest titers at day two after inoculation. at day four post-inoculation a significant reduction of the viral titers was observed. in the case of animals infected with or pfu, viremia was not detected or only detected in one of the three infected mice, respectively ( figure b ). overall, these results indicated that the rzikv-rgn recovered from the infectious clone is virulent in mice but only at high ( pfu) dose. week-old a mice (five mice per group) were mock-infected (pbs) or infected s.c. in the footpad with the indicated pfu of rzikv-rgn, and body weight loss (expressed as the percentage of starting weight, left panel) and survival (right panel) were monitored daily during days. mice that lost more than % of their initial body weight or presented hind limb paralysis were humanely euthanized. error bars represent standard deviations of the mean for each group of mice. asterisks indicate that the differences between viral doses of and are statistically significant when data are compared using the unpaired t test (*, p < . ; **, p < . ). (b) viral titers in mice sera. female -to- -week-old a mice (six mice per group) were infected with the indicated pfu of rzikv-rgn as described above, and viral titers in sera were determined at days two and four after infection (three animals per time point) by plaque assay and immunostaining using the pan-flavivirus e protein mab g . symbols represent data from individual mice and bars the geometric means of viral titers. asterisks indicate that the differences in viral titers between experimental samples are statistically significant when data are compared using the unpaired t test (**, p < . ; ***, p < . ). &: virus not detected in two mice; nd: virus not detected. the detection limit of the assay ( pfu/ml) is indicate as a dashed line. during the assembly of the pbac-zikv-rgn infectious clone, we detected the presence of a point mutation in the ns a protein, which was introduced during the chemical synthesis of fragment zikv . this mutation consists in a cytosine-to-thymidine substitution at genomic position , resulting in an alanine-to-valine change in the residue of the ns a protein (a v). because this mutation consists of a conservative amino acid change, we decided to explore the possibility of using this mutation as a genetic marker. to this end, the infectious clone pbac-zikv-rgn-mns a was generated by replacing the zikv wt fragment for that containing the ns a a v mutation. this infectious clone was fully stable in bacteria and no additional mutations were observed after sequencing the full-length clone. after that, vero cells were transfected with the mutant infectious clone and the recovery efficiency of the rzikv-rgn-mns a mutant virus was compared to that of the parental rzikv-rgn virus ( figure ). although the infectious virus was recovered in both cases, virus production was one logarithm lower in the case of the mutant rzikv-rgn-mns a, reaching maximum titers of pfu/ml at seven days post-transfection ( figure a ). when the plaque phenotype was analyzed, we found that the plaque size of the mutant rzikv-rgn-mns a was smaller (more than a -fold reduction) than that of the parental rzikv-rgn ( figure b ), indicating that the a v mutation, despite of being a conservative substitution, caused reduction in plaque size and virus production. in addition, an in silico analysis was performed to evaluate the frequency of amino acid residues of the ns a protein in more than zikv strains sequences deposited in the database [ ] (https://www.viprbrc.org/brc/home.spg?decorator=flavi). this analysis indicated that amino acid a is highly conserved, since the % of the analyzed zikv sequences contained an alanine residue at this position. to further confirm the effect of the ns a a v mutation on virus production, the growth kinetics at high ( pfu/cell) and low ( . pfu/cell) moi of the mutant virus were compared to those of the parental virus ( figure c ). again, a reduction of about one logarithmic unit in virus production was detected in vero cells infected with the mutant virus both at high and low moi ( figure c ). taken into consideration that flavivirus ns a protein is involved in regulation of rna replication and virus assembly [ ] , we further analyzed whether the reduction in plaque size and virus production of the mutant virus was associated with reduced viral rna synthesis. to this end, the production of viral rna in vero cells infected with either the parental or mutant viruses at an moi of . pfu/cell was analyzed at and hpi by rt-qpcr using a custom taqman assay specific for zikv-rgn genome ( figure d ). at both times, a -fold reduction in the levels of viral rna was observed in cells infected with the mutant virus ( figure d ), confirming that ns a a v mutation at least impairs viral rna synthesis. in agreement with these data, a reduction in the expression levels of zikv e protein was observed by ifa in vero cells infected with the mutant virus in comparison to cells infected with the parental virus ( figure e ). finally, to discard the presence of other undesired mutations, the full-length sequence of the mutant virus was determined by deep-sequencing, and no mutations other than ns a a v were detected. collectively, these results indicated that ns a a v mutation alone affected zikv growth in vero cells at least by impairing viral rna synthesis. to investigate whether the reduced rna synthesis of rzikv-rgn-mns a in vero cells could result in viral attenuation in vivo, the ability of the mutant virus to induce pathogenesis was analyzed in a mice and compared with that of the parental rzikv-rgn ( figure ). to that end, groups of five female -to- -week-old a mice were inoculated s.c. in the footpad with pfu of either rzikv-rgn or rzikv-rgn-mns a, or with pbs as a negative control, and the body weight loss and survival were monitored daily over days. in contrast to mice infected with rzikv-rgn that quickly lost weight and all of them died at day eight after infection, mice infected with the mutant rzikv-rgn-mns a did not presented any clinical signs of infection or weight loss and all of them survived to viral infection ( figure a ). to further analyze the correlation of the attenuation of the mutant virus with viral replication, presence of the virus in mice sera was analyzed at days two and four post-infection. in agreement with the pathogenicity data, mice infected with the mutant virus presented lower viremia than mice infected with the parental virus ( figure b ). the mutant virus was only detected at day two after infection and at lower titers (approximately . logarithms lower) than the parental virus. as an internal control of the experiment, the plaque phenotype of the viruses recovered from the blood of infected mice were analyzed. as expected, rzikv-rgn formed big plaques while the mutant rzikv-rgn-mns a formed small plaques ( figure c ). these results indicated that rzikv-rgn-mns a was highly attenuated in mice, as compared to rzikv-rgn, and that this attenuation may be due to a lower replication of the rzikv-rgn-mns a mutant virus. after elucidating that rzikv-rgn-mns a was attenuated in vivo, its ability to induce protection against a challenge with the parental rzikv-rgn was analyzed (figure ) . to that end, groups of five female -to- -week-old a mice were vaccinated s.c. in the footpad with pfu of rzikv-rgn-mns a or mock-vaccinated with pbs. twenty days after vaccination, blood samples were collected to evaluate the humoral response. one day later, mice were challenged with a lethal dose ( pfu) of rzikv-rgn and the body weight loss and survival were analyzed daily over figure . pathogenesis of rzikv-rgn-mns a in a mice. (a) weight loss and mortality. female -to- -week-old a mice (five mice per group) were mock-infected (pbs) or infected s.c. in the footpad with pfu of rzikv-rgn or rzikv-rgn-mns a, and body weight loss (expressed as the percentage of starting weight, left panel) and survival (right panel) were monitored daily during days. mice that lost more than % of their initial body weight or presented hind limb paralysis were humanely euthanized. error bars represent standard deviations of the mean for each group of mice. (b) viral titers in mice sera. female -to- -week-old a mice (six mice per group) were infected with pfu of rzikv-rgn (wt) or rzikv-rgn-mns a (mut) as described above, and viral titers in sera were determined at days two and four after infection (three animals per time point) by plaque assay and immunostaining using the pan-flavivirus e protein mab g . symbols represent data from individual mice and bars the geometric means of viral titers. asterisks indicate that the differences between rzikv-rgn and rzikv-rgn-mns a are statistically significant when data are compared using the unpaired t test (***, p < . ). nd: virus not detected. the detection limit of the assay ( pfu/ml) is indicate as a dashed line. (c) plaque phenotype. vero cells at % confluence ( -well plate format) were infected with pfu of rzikv-rgn (left) or rzikv-rgn-mns a (right) recovered from infected mice at day two post-infection and the plaque size evaluated by plaque assay and immunostaining using the pan-flavivirus e protein mab g . after elucidating that rzikv-rgn-mns a was attenuated in vivo, its ability to induce protection against a challenge with the parental rzikv-rgn was analyzed (figure ) . to that end, groups of five female -to- -week-old a mice were vaccinated s.c. in the footpad with pfu of rzikv-rgn-mns a or mock-vaccinated with pbs. twenty days after vaccination, blood samples were collected to evaluate the humoral response. one day later, mice were challenged with a lethal dose ( pfu) of rzikv-rgn and the body weight loss and survival were analyzed daily over days. as expected, mice vaccinated with pbs lost weight rapidly, showed clear symptoms of disease, and all of them succumbed to challenge with rzikv-rgn. in contrast, mice vaccinated with rzikv-rgn-mns a did not lose weight and all of them survived the challenge with rzikv-rgn ( figure a ), indicating that a single immunization dose with rzikv-rgn-mns a is enough to induce full protection against zikv-rgn. in agreement with these data, a strong humoral response against zikv-rgn was observed in mice vaccinated with rzikv-rgn-mns a ( figure b ) and no viremia was detected in sera samples of vaccinated mice at days two and four after challenge ( figure c ). figure a ), indicating that a single immunization dose with rzikv-rgn-mns a is enough to induce full protection against zikv-rgn. in agreement with these data, a strong humoral response against zikv-rgn was observed in mice vaccinated with rzikv-rgn-mns a ( figure b ) and no viremia was detected in sera samples of vaccinated mice at days two and four after challenge ( figure c ). once confirmed that rzikv-rgn-mns a was attenuated in vivo and induces protection against zikv-rgn in mice, the genetic stability of the mutant virus was analyzed in vero cells, in order to test the possible use of this mutant virus as a base for the development of a live-attenuated zikv vaccine (figure ). to this end, both mutant (rzikv-rgn-mns a) and parental (rzikv-rgn) viruses were passaged five times in vero cells (p to p ) and the virus plaque phenotype, growth kinetics and the sequence of ns a were analyzed for each passage. analysis of the plaque phenotype showed that the parental virus presented the expected plaque size throughout all passaging. in contrast, for the mutant virus a reversion to parental plaque phenotype was observed. this reversion started at p ( % of big plaques), clearly increased at p ( % of big plaques) and was complete at p ( figure a ). after that, we analyzed whether this plaque size reversion of the mutant rzikv-rgn-mns a correlated with an increase in virus replication to levels of that of the parental virus. to that end, the growth kinetics (moi of pfu/cell) of the mutant virus from p and p were compared to those of the parental virus. growth curve analysis showed that in contrast to the mutant virus from p , the virus from p replicated to the same levels as the parental virus ( figure b ), suggesting that the mutant virus reverted to the wt sequence during its propagation in vero cells. to confirm these observations, the ns a coding region of mutant viruses from p to p was amplified by rt-pcr and sequenced. sequence analysis confirmed the reversion of the a v mutation to the wt sequence. although the instability and reversion of the mutant virus to the wt sequence during its propagation in vero cells limits the use of this mutant for vaccine development, these data further support the importance of this ns a residue for virus replication. at hpi, cell culture supernatants were collected and used to infect fresh vero cells. this process was repeated four more times and virus stocks of passages to (p to p ) were generated. (a) plaque size. vero cells were infected with the different passages (p to p ) of rzikv-rgn or rzikv-rgn-mns a, and at four days post-infection the viral plaques were visualized by immunostaining using the pan-flavivirus e protein mab g . (b) growth kinetics. vero cells at % confluence ( -well plate format; triplicates) were infected (moi of pfu/cell) with p and p of rzikv-rgn or rzikv-rgn-mns a and at the indicated hpi, virus titers were determined by plaque assay. error bars represent standard deviations of the mean from three experiments. asterisks indicate that the differences between rzikv-rgn-mns a p and the experimental samples, rzikv-rgn p , rzikv-rgn p and rzikv-rgn-mns a p , are statistically significant when data are compared using the unpaired t test (***, p < . ). analysis of the plaque phenotype showed that the parental virus presented the expected plaque size throughout all passaging. in contrast, for the mutant virus a reversion to parental plaque phenotype was observed. this reversion started at p ( % of big plaques), clearly increased at p ( % of big plaques) and was complete at p ( figure a ). after that, we analyzed whether this plaque size reversion of the mutant rzikv-rgn-mns a correlated with an increase in virus replication to levels of that of the parental virus. to that end, the growth kinetics (moi of pfu/cell) of the mutant virus from p and p were compared to those of the parental virus. growth curve analysis showed that in contrast to the mutant virus from p , the virus from p replicated to the same levels as the parental virus ( figure b ), suggesting that the mutant virus reverted to the wt sequence during its propagation in vero cells. to confirm these observations, the ns a coding region of mutant viruses from p to p was amplified by rt-pcr and sequenced. sequence analysis confirmed the reversion of the a v mutation to the wt sequence. although the instability and reversion of the mutant virus to the wt sequence during its propagation in vero cells limits the use of this mutant for vaccine development, these data further support the importance of this ns a residue for virus replication. moreover, these data also suggest that zikv ns a protein represents a good target for the development of antivirals against zikv infection. the significance of zikv to public health due its association with guillain-barré syndrome and fetal abnormalities [ ] [ ] [ ] [ ] [ ] [ ] , together with the lack of approved antiviral agents or vaccines, have triggered a global effort to study this flavivirus in order to develop effective strategies to prevent and control zikv infection in humans. in this respect, the development and implementation of reverse genetic approaches for zikv provide investigators with a novel and powerful experimental tool to study both the biology and pathogenesis of zikv as well as the development of attenuated forms of zikv for their implementation as live-attenuated vaccines. however, as described for other flaviviruses, the generation of zikv infectious clones using traditional approaches are very difficult due to the toxicity and instability of some viral sequences when they were propagated as cloned cdna in bacteria [ ] [ ] [ ] . in the past two years, several approaches that overcomes this toxicity problem have been applied for the successfully generation of zikv infectious clones. these include the use of low-copy plasmids [ , ] , insertion of introns to disrupt toxic sequences [ ] [ ] [ ] , mutational silencing of cbps present in the viral genome [ ] , in vitro ligation of cdna fragments [ , , ] , the isa method [ , ] , and the cper approach [ ] . although very useful, some of these approaches are laborious, time consuming and present several disadvantages. for instance, most of them need in vitro ligation and transcription steps that complicate the assembly and reduce the recovery efficiency. moreover, these low recovery efficiencies increase the presence of undesired mutations that could result in in vitro and/or in vivo attenuation, limiting the use of these infectious clones for certain studies. others, such as the mutational silencing of cbps, in which a high number of silent mutations have to be introduced, could affect viral fitness. finally, the use of low-copy plasmids has been shown to be effective for several zikv strains but not for others, probably due the different degrees of toxicity of rna sequences of different strains [ , , , ] . here, we describe a powerful approach for the generation of an infectious cdna clone of the zikv-rgn strain in a single plasmid, based on the use of a combination of synthetic biology and bacs. the full-length cdna copy of the zikv-rgn strain was generated from four synthetic dna fragments and cloned in the bac plasmid pbelobac [ ] under the control of the cmv promoter, which allows the expression of the viral rna in the nucleus [ ] , and flanked at the '-end by the hdv ribozyme and the bgh polyadenylation and termination sequences to produce synthetic rnas bearing authentic '-ends of the viral genome. the bac cdna clone was fully stable during its propagation in bacteria and the functional infectious virus was rescued after direct transfection of susceptible vero cells that was pathogenic in a mice. a zikv-rgn infectious clone generated using the cper approach has been recently reported [ ] . however, in contrast to our results, the rescued virus was asymptomatic and nonlethal in female -to- -week-old a mice infected with doses of to ccid ( % cell culture infective doses) via the s.c. route. whether the differences in pathogenicity among this rzikv-rgn and ours are related to the experimental approach (cper versus bac), the age of the mice ( -to- -week-old versus -to- -week-old) or the moi used to infect the mice ( - ccid versus pfu) remain to be evaluated. although other zikv reverse genetic systems have been reported (discussed above), the bac approach constitutes an useful alternative that presents important advantages: (i) the bac plasmids present a strictly controlled replication leading to only one plasmid per cell and therefore minimize the toxicity associated with several flavivirus sequences when amplified in bacteria [ ] . this allows the easy and direct manipulation of the viral genome for molecular studies; (ii) similarly to other approaches using polii-driven promoters, the bac approach results in intracellular expression of the viral rna [ , , , , ] , allowing the capping of the viral rna and the recovery of infectious virus without the need of an in vitro transcription step. although some splicing events could occur during the nuclear expression of the viral genome, mainly due to the presence of donor and acceptor putative sequences in the viral genome, the efficiency of this phenomenon is very low and does not affect the recovery of infectious viruses [ ] ; (iii) like other systems based on transfection of dna constructs [ , , , , ] , bac cdna clones present a higher efficiency of transfection than rna transcripts in mammalian cells. this allows higher efficiencies of virus recovery, reducing the passages in cell culture to get a viral stock and therefore, the possibility of introducing undesired mutations by cell culture adaptation; (iv) the manipulation of bac cdna clones is relatively easy and similar to that of conventional plasmids with slight modifications due to the presence of only one plasmid copy per cell. in addition to standard protocols, the bac cdna clones could also be efficiently modified into e. coli by homologous recombination using a two-step approach that combine the red recombination system and counterselection with the homing endonuclease i-scei [ ] [ ] [ ] [ ] ; and (v) the bac approach has been successfully used to engineer infectious clones of other flaviviruses, including denv [ ] , and several coronaviruses that contain the largest viral rna genome known and similar toxicity problems to those described for flaviviruses [ , [ ] [ ] [ ] [ ] . these data highlight the potential of the bac approach for the rapid and reliable construction of stable infectious clones of emerging flavivirus and other similar rna viruses with unstable viral genomes when amplified as cdnas in bacteria. the zikv reverse genetic system described in this article was further used to study the effect of a single amino acid substitution (a v) in the viral ns a protein on virus growth in cultured cells and pathogenesis in vivo. our results suggested that this single amino acid change impaired viral rna synthesis and virus production in cell culture and highly attenuated the virus in mice. however, we cannot discard that this mutation in the ns a protein could affect other steps in the replication cycle of the virus. flavivirus ns a protein is a -kda hydrophobic protein associated with the endoplasmic reticulum that contains eight transmembrane domains. it is a multifunctional protein that has been involved in viral rna synthesis [ , ] , virus assembly [ , ] , membrane rearrangement [ ] , and immunomodulation of innate immune response [ ] [ ] [ ] . by homology with the denv ns a topology, the zikv ns a a v mutation maps in the last transmembrane domain, for which no specific function has been reported. therefore, our data constitute the first evidence of a role of this ns a domain in viral rna synthesis. on the other hand, it is important to note that the ns a a v mutation promoted a -fold reduction in viral rna synthesis and more than -fold reduction in virus production. this reduced virus production could be a consequence of the rna synthesis impairment. however, since the reduction in virus production is higher than that observed in rna synthesis and that flavivirus ns a protein is also involved in virus assembly, we cannot discard an additional effect of a v mutation in zikv assembly. in addition, we have found that the mutant virus was attenuated in a mice. this attenuation could be explained as a consequence of the lower rna synthesis of the mutant virus. however, an additional effect of the a v mutation on the putative immunomodulatory role of ns a protein [ ] [ ] [ ] , leading to virulence attenuation, cannot be discarded. future studies will be required to determine whether this mutation affects only viral rna synthesis or also virus assembly and immunomodulation of the host defenses. importantly, we have shown that immunization with a single dose of pfu of the mutant rzikv-rgn-mns a induced protection against a lethal challenge with the parental rzikv-rgn, suggesting the potential implementation of this ns a mutant as the base of a live-attenuated vaccine. unfortunately, the mutant rzikv-rgn-mns a was instable and reverted to the wt sequence during its propagation in vero cells, limiting the use of this mutation alone for vaccine development. however, this instability and the high conservation of the amino acid a of the ns a protein among zikv strains highlights the importance of this ns a residue for virus replication, and therefore the potential use of ns a protein as a good target for antiviral development against zikv infection. in summary, we have developed a powerful zikv reverse genetic system based on the use of bacs that has allowed us to identify a single point mutation in the ns a protein that attenuates the virus in vitro and in vivo. this infectious clone system provides a valuable tool to the research community to explore zikv molecular biology, viral determinants of zikv pathogenesis, virus-host interactions, and vaccine and antivirals developments. an update on zika virus infection who calls off global zika emergency the . å resolution cryo-em structure of zika virus probing molecular insights into zika virus(-)host interactions zika virus: a report on three cases of human infection during an epidemic of jaundice in nigeria simultaneous outbreaks of dengue, chikungunya and zika virus infections: diagnosis challenge in a returning traveller with nonspecific febrile illness the global threat of zika virus to pregnancy: epidemiology, clinical perspectives, mechanisms, and impact zika virus outbreak on yap island, federated states of micronesia zika virus, french 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information for publication of quantitative real-time pcr experiments an open-source platform for biological-image analysis canine influenza viruses with modified ns proteins for the development of live-attenuated vaccines a susceptible mouse model for zika virus infection a mouse model of zika virus pathogenesis characterization of a novel murine model to study zika virus committee for the update of the guide for the care and use of laboratory animals sindbis virus dna-based expression vectors: utility for in vitro and in vivo gene transfer virus pathogen resource (vipr), faviviridae. available online engineering the largest rna virus genome as an infectious bacterial artificial chromosome targeted modification of a human β-globin locus bac clone using get recombination and an i-scei counterselection cassette a highly efficient escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of bac dna two-step red-mediated recombination for versatile high-efficiency markerless dna manipulation in escherichia coli a new logic for dna engineering using recombination in escherichia coli construction of a severe acute respiratory syndrome coronavirus infectious cdna clone and a replicon to study coronavirus rna synthesis engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate molecular characterization of feline infectious peritonitis virus strain df- and studies of the role of orf abc in viral cell tropism recovery of a neurovirulent human coronavirus oc from an infectious cdna clone subcellular localization and some biochemical properties of the flavivirus kunjin nonstructural proteins ns a and ns a mutations in west nile virus nonstructural proteins that facilitate replicon persistence in vitro attenuate virus replication in vitro and in vivo mutations in the yellow fever virus nonstructural protein ns a selectively block production of infectious particles role of nonstructural protein ns a in flavivirus assembly analysis of adaptive mutations in kunjin virus replicon rna reveals a novel role for the flavivirus nonstructural protein ns a in inhibition of β interferon promoter-driven transcription a single amino acid substitution in the west nile virus nonstructural protein ns a disables its ability to inhibit α/β interferon induction and attenuates virus virulence in mice subversion of interferon by dengue virus we are grateful to carla gómez and snezhana dimitrova for technical assistance in the bac clone generation and mice experiments, respectively. we also thank sylvia gutiérrez and ana oña at the cnb advanced microscopy facility for their valuable support in immunofluorescence microscopy analysis. the authors declare no conflict of interest. the funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, and in the decision to publish the results. key: cord- - syrrn authors: yang, yong-le; qin, pan; wang, bin; liu, yan; xu, guo-han; peng, lei; zhou, jiyong; zhu, shu jeffrey; huang, yao-wei title: broad cross-species infection of cultured cells by bat hku -related swine acute diarrhea syndrome coronavirus and identification of its replication in murine dendritic cells in vivo highlight its potential for diverse interspecies transmission date: - - journal: j virol doi: . /jvi. - sha: doc_id: cord_uid: syrrn outbreaks of severe diarrhea in neonatal piglets in guangdong, china, in resulted in the isolation and discovery of a novel swine enteric alphacoronavirus (seacov) derived from the species rhinolophus bat coronavirus hku (y. pan, x. tian, p. qin, b. wang, et al., vet microbiol : – , ). seacov was later referred to as swine acute diarrhea syndrome cov (sads-cov) by another group (p. zhou, h. fan, t. lan, x.-l. yang, et al., nature : – , ). the present study was set up to investigate the potential species barriers of sads-cov in vitro and in vivo. we first demonstrated that sads-cov possesses a broad species tropism and is able to infect cell lines from diverse species, including bats, mice, rats, gerbils, hamsters, pigs, chickens, nonhuman primates, and humans. trypsin contributes to but is not essential for sads-cov propagation in vitro. furthermore, c bl/ j mice were inoculated with the virus via oral or intraperitoneal routes. although the mice exhibited only subclinical infection, they supported viral replication and prolonged infection in the spleen. sads-cov nonstructural proteins and double-stranded rna were detected in splenocytes of the marginal zone on the edge of lymphatic follicles, indicating active replication of sads-cov in the mouse model. we identified that splenic dendritic cells (dcs) are the major targets of virus infection by immunofluorescence and flow cytometry approaches. finally, we demonstrated that sads-cov does not utilize known cov receptors for cellular entry. the ability of sads-cov to replicate in various cells lines from a broad range of species and the unexpected tropism for murine dcs provide important insights into the biology of this bat-origin cov, highlighting its possible ability to cross interspecies barriers. importance infections with bat-origin coronaviruses (covs) (severe acute respiratory syndrome cov [sars-cov] and middle east respiratory syndrome cov [mers-cov]) have caused severe illness in humans after “host jump” events. recently, a novel bat-hku -like cov named swine acute diarrhea syndrome cov (sads-cov) has emerged in southern china, causing lethal diarrhea in newborn piglets. it is important to assess the species barriers of sads-cov infection since the animal hosts (other than pigs and bats) and zoonotic potential are still unknown. an in vitro susceptibility study revealed a broad species tropism of sads-cov, including various rodent and human cell lines. we established a mouse model of sads-cov infection, identifying its active replication in splenic dendritic cells, which suggests that sads-cov has the potential to infect rodents. these findings highlight the potential cross-species transmissibility of sads-cov, although further surveillance in other animal populations is needed to fully understand the ecology of this bat-hku -origin cov. different animal species were tested for susceptibility to sads-cov treated with or without trypsin ( table ) . as a brief summary of the results, of the cell lines showed significant susceptibility to sads-cov infection, defined by efficient viral replication, antigen expression, and the appearance of cytopathic effect (cpe). the three cell lines that were not infected by sads-cov were mdck, bfk, and raw . . first, cpe was examined by inverted light microscopy at hours postinfection (hpi), and scores are shown in table . as the t, nih/ t , cho, brl- a, and nrk- e cell lines were sensitive to trypsin, they could not be tested for sads-cov infection in mmt cells. apart from that, cpe was visible in vero, st, and brl- a cell lines without trypsin, and prominent cpe appeared in or was enhanced with trypsin in marc- , cos- , bsc- , vero, st, pk , llc-pk , and bhk- cell lines ( table ) . as some cells did not display cpe after sads-cov infection, all cell lines were subsequently tested for viral m protein expression by immunofluorescence assay (ifa) fig. ) , revealing the same range as seen by cpe in the different cell lines (data not shown). syncytium formation was prominent in huh- , vero, and bhk- cells, whereas in mdck, bfk and raw . cells, the antigen expression was much less prominent than in the other cell lines (fig. a , c, and i). most cell lines tested showed evidence of productive infection, as indicated by expression of the m protein, while the inefficient antigen expression in marc- , llc-pk , and ipec-j cells suggested only a limited infection. next, viral load in the culture supernatants was detected over days postinfection (dpi) by reverse transcriptase quantitative pcr (qrt-pcr) ( fig. a to c). a higher mean viral load was detected by qrt-pcr after trypsin treatment in hepg , hela, marc- , cos- , bsc- , vero, llc-pk , ipec-j , bhk- , and df- cells. therefore, trypsin contributes to but is not essential for sads-cov propagation in these cell lines. there was no difference after trypsin treatment in the other cell lines, although huh- and tb- cells had high levels of sads-cov rna regardless of trypsin treatment. the progressive release of infectious sads-cov into the culture medium of six representative cell lines infected with sads-cov was determined by titration of super- to determine the effect of trypsin on sads-cov infection, each cell line was infected in the following three conditions: "no trypsin" treatment, inoculated with sads-cov diluted in maintenance medium (mm) for h and subsequently replaced with mm (a); "pretrypsin" treatment, inoculated with sads-cov diluted in mm containing g/ml trypsin (mmt) for h and subsequently replaced with mm (b); and "double-trypsin" treatment, inoculated with sads-cov in mmt and subsequently replaced with mmt (c). infection supernatants were collected at , , , , , and hpi for viral load detection by a qrt-pcr assay targeting the viral n gene. data are expressed as the mean viral load (log copies/l) Ϯ standard deviation (sd), and all experiments were performed in triplicate. the t, cho, brl- a, and nrk- e cell lines did not survive in the presence of trypsin. (d) infectious titers (tcid /ml) of sads-cov secreted from hela, vero, tb- , bhk- pk- , and mdck cells were determined on vero cells. natants in vero cells (fig. d ). unlike in mdck cells, sads-cov infection of hela, vero, tb- , bhk- , and pk- cells was productive, with hela cells showing the greatest susceptibility (fig. d) . wild-type c bl/ j mice can be infected by sads-cov via oral and intraperitoneal routes. with the observation that sads-cov could infect diverse rodent cell lines (from mice, rats, and hamsters as well as gerbil primary kidney cells), we hypothesized that mice may be susceptible to sads-cov. to test this, we inoculated -to -week-old wild-type b mice with ϫ % tissue culture infective dose (tcid ) of sads-cov by the per oral (p.o.) or intraperitoneal (i.p.) route and monitored them for days for clinical symptoms. the mice did not succumb to the infection nor did they develop diarrhea or experience weight loss during the incubation period (data not shown). to determine whether sads-cov infected the animals asymptomatically, tissue and fecal samples from inoculated mice were collected at , , , , , , and dpi to determine viral growth kinetics and shedding. analysis of tissue samples by qrt-pcr suggested that sads-cov replicated modestly in the stomach early after i.p. or p.o. infection, declining and reaching undetectable levels at or dpi and thereafter (fig. a) . a very limited viral replication was observed in each region of the small intestine, with the ileum via i.p. infection showing continuous and decent detectable viral rna (fig. b ). in the large intestine, i.p. infection also resulted in viral rna loads slightly above the limit of detection at each time point in the ceca, whereas it led to higher viral rna levels at to dpi, and much lower viral rna at to dpi in the colon than that of the p.o. route (fig. c) . however, this replication in the large intestine did not translate into higher shedding, as hardly any viral genomes were detected, even at dpi in the fecal samples collected from i.p.-infected mice (fig. f ). on the contrary, significantly more virus was detected in the feces of p.o.-infected mice at and dpi, indicating that i.p. inoculation does not lead to higher virus shedding. finally, sads-cov replicated more efficiently in the spleen following the i.p. route, with significantly higher viral rna loads at dpi (fig. d ). more importantly, the virus was not cleared from this tissue by dpi in the i.p.-infected group and by dpi in the p.o.-infected group, suggestive of a sads-cov prolonged infection in the spleen independent of inoculation route. in contrast to the spleen, only very low levels of viral rna were detected in the local lymphoid tissue of mesenteric lymph nodes (mlns) at to dpi, and no virus was detectable at later time points (fig. d ). we also looked for virus in other extraintestinal sites, including the heart, lungs, liver, kidneys, and blood, but they were all negative or had extremely low levels (fig. e ). igg antibody levels after days detected by sads-cov virion-based enzyme-linked immunosorbent assay (elisa) showed that the i.p. route could effectively elicit host immune responses (fig. g) . splenocytes support sads-cov replication. with the mouse infection model described above, our next step was to determine the cell tropism of sads-cov in vivo. thus, we performed immunohistochemistry (ihc) on sections of small and large intestine and spleen from mice infected i.p. with ϫ tcid of sads-cov at dpi. a monoclonal antibody against double-stranded rna (dsrna) was used to identify cells that supported active virus replication, as dsrna is an intermediate that only exists during intracellular viral replication. sads-cov dsrna signals were observed in the splenic white pulp in the marginal zone on the edge of lymphatic follicles and in the margins of the periarteriolar lymphocyte sheath (fig. a ). staining of tissue sections from mock-infected mice were used as a control (fig. b ). in addition to dsrna, we also used rabbit polyclonal antibodies (pabs) to detect the expression of the viral structural protein (m) or nonstructural protein (nsp -ac). at dpi, anti-m or anti-ac staining was observed in the white pulp around the lymphatic nodules (fig. c) , similar to the localization of dsrna staining (fig. a ). tissue sections from sads-cov or mockinfected mice probed with preimmune sera were negative, indicating the specificity of the sads-cov antibody. unfortunately, neither viral proteins (structural or nonstruc-tural) nor dsrna were detected in the intestine of infected mice, consistent with the detection of only very low levels of viral rna in these tissues by qrt-pcr (fig. ) . next, sads-cov infection was quantified in the spleen using flow cytometry. we inoculated b wild-type mice with ϫ tcid of virus either i.p. or p.o. and extracted the bulk immune cells from the spleen of infected animals at dpi. the flow cytometry method was first validated in vero cells infected with sads-cov at a multiplicity of infection (moi) of . , followed by staining with a pab against the n or ac protein at hpi (fig. d ). as the anti-ac pab exhibited optimal intracellular staining for viral signals (fig. d) , it was used to determine the percentage of infected splenocytes. there were approximately . -and . -fold increases of total splenocytes positive for virus replication after p.o. and i.p. inoculation, respectively (fig. e , left; fig. f ), with a significant increase in the total number of ac-positive splenocytes in i.p.-infected mice compared with that of p.o. (fig. e , right). these data are consistent with the significantly lower viral loads in the spleen at and dpi in p.o.-inoculated mice (fig. d ), suggesting better virus dissemination and replication and escape from mucosal immune clearance. we then evaluated the growth characteristics of sads-cov in splenocytes by assessing antigen production and replication kinetics ex vitro. splenocytes were first extracted from naive mice, plated in -mm dishes, and infected with ϫ tcid of sads-cov. we observed clusters of infected cells that appeared to have been engulfed by phagocytes (fig. g , middle), and the structural n protein was shown in the cytoplasm of infected cells by confocal microscopy (fig. g , middle and right). the percentage of infected cells was quantified by flow cytometry using anti-ac pab, revealing a nearly -fold increase in the splenocytes positive for viral signals (fig. h) , very similar to the percentage of infection observed in vivo. to further characterize the growth kinetic of sads-cov in primary splenocytes, cells were infected with ϫ tcid of sads-cov, and culture supernatants were harvested at , , , , and hpi. active viral replication was confirmed, with a . -log time-dependent increase in genomic rna equivalents, plateauing from to hpi (fig. i ). these data suggest that although only ϳ % of splenocytes were infected, and these cells supported a decent level of viral replication. together, these results indicate that sads-cov productively infects mouse splenocytes. splenic dcs support sads-cov replication. splenocytes were harvested from i.p.-infected mice at dpi, and the extracted cells were costained with antibodies against sads-cov-ac and each of four cell surface markers (anti-cd for b cells, anti-cd for t cells, anti-cd /c ϩ for dcs, and anti-f / ϩ for macrophages) using flow cytometry (fig. a ). the percentage of infected cd /c ϩ cells was significantly higher than the other cell subgroups, indicating that dcs are the major targets of sads-cov infection in the spleen. the phenotype was further confirmed by double-staining ifa with anti-dsrna, anti-m, or anti-ac antibody plus anti-cd /c ϩ in splenic sections. as expected, dsrna staining overlapped with the cd /c surface marker on the edges of lymphatic follicles (fig. b) , whereas no viral signals were seen in the mock-infected control (fig. c ). similar patterns of costaining were detected by m and ac antibodies (fig. d ). to gain insight into the relative quantity of dcs compared with other undefined target cells, cells positive for dsrna and cd /c were counted in to different microscope fields of spleens from infected mice (fig. e) , showing that . % of sads-covinfected cells were dcs (fig. f ). to our knowledge, these results reveal the most extensive cell tropism among known covs, suggesting the functional receptor(s) for sads-cov is likely to be a very common molecule. in order to test this hypothesis, it was first necessary to find a cell line that was refractory to infection only at the internalization step. mdck cells, which showed undetectable virus production in early infection tests ( fig. and ) , were chosen as a potential candidate. there are four known types of functional cov protein receptors, including angiotensin converting enzyme (ace ) for sars-cov ( ), dipeptidyl peptidase (dpp ) for mers-cov ( ) , aminopeptidase n (apn) for tgev ( ) and pdcov ( , ) , and mouse carcinoembryonic antigen-related cell adhesion molecule a (mceacam a) for mouse hepatitis virus (mhv) ( ) . to test whether one of these molecules serves as the sads-cov receptor, we attempted to inoculate nonsusceptible mdck cells overexpressing porcine apn, human dpp , mouse ceacam a, or human ace with sads-cov, but none of them allowed infection, as staining with anti-sads-cov-n pab was negative (fig. a) . meanwhile, the expression of each receptor in mdck cells was confirmed by ifa (fig. a) and western blot analysis (fig. b) using antibodies against the tags fused to the receptors. we confirmed that, as positive controls, lentiviruses pseudotyped with tgev, sars-cov, mers-cov, or mhv spike (i.e., pseudoviruses) efficiently entered mdck cells exogenously expressing the respective receptors (fig. c) . next, we demonstrated that mdck cells can confer sads-cov replication competency by transfection of a sads-cov/seacov infectious cdna clone established recently ( ) , as simultaneous expression of nsp -ac and n proteins were clearly detected by ifa (fig. d) . moreover, passaging of supernatants from psea-transfected mdck cells onto fresh vero cells resulted in progeny sads-cov infection, as evidenced by expression of the n protein (fig. e) , indicating that mdck cells can also support infectious sads-cov production without cell-to-cell spread. therefore, sads-cov apparently does not utilize any of the known cov receptors for cellular entry. the same conclusion was reached using hela cells overexpressing each of the four classical cov receptors followed by sads-cov inoculation by zhou et al. ( ) ; however, the hela cell line itself was most susceptible to sads-cov infection in the present study (fig. d) . in order to assess the potential species barriers of sads-cov infection, a cell line susceptibility study was first conducted using different cell lines. as sads-cov probably originated from a bat sadsr-cov ( ) derived from hku -cov identified in rhinolophus sinicus (chinese horseshoe bats) ( ), we commenced testing viral susceptibility in two available bat cell lines, namely, bfk from myotis daubentonii ( ) and tb- from tadarida brasiliensis. although bfk cells did not support sads-cov replication, it replicated efficiently in tb- cells (fig. a and fig. ) , suggesting that other bat species in addition to horseshoe bats are likely susceptible to sads-cov infection. interestingly, sads-cov protein expression was detected in almost all of the rodent cells (hamster, gerbil, mouse, and rat) including bhk- , which is not susceptible to other known human covs, such as sars-cov and mers-cov ( , ) , as well as three swine enteric covs, namely, pedv, pdcov, and tgev ( ) . given the fact that sads-cov infects both primary and passaged or primary cell lines originating from rodents, we hypothesized that rodents may be susceptible to sads-cov infection. to explore this possibility, we challenged wild-type b mice with sads-cov by two different inoculation routes. the challenged animals neither succumbed to infection nor manifested any signs of gastroenteritis. in fact, experimental infection of neonatal piglets with a higher dose of purified sads-cov in our laboratory resulted only in mild diarrheal signs or subclinical infection ( ) . also, there was a lack of robust viral replication in the intestines during infection, and no tissue damage was detected throughout the intestines ( fig. b and c) , reflecting the suboptimal infection by sads-cov in immunocompetent wild-type mice. on the contrary, the virus had more efficient replication within the spleen, which was reflected by a continuous detection of viral genomic rna in the immune cells at all time points over a -day period (fig. d) . the phenotype was also consistent with the replication kinetics in extracted splenocytes in vitro, in which viral genomic rna peaked and plateaued at hpi ( fig. g and i) . these data collectively led to speculation that sads-cov favors splenic cells over other tissues. the most logical explanation for these tissue-specific discrepancies in virus replication is (i) target cells are more concentrated in the spleen and more sporadic in the intestine or (ii) splenic immune cells have enhanced expression of the unknown receptor(s) over intestinal cells. the animals were more susceptible to i.p. infection, resulting in higher virus replication in the distal section of the small intestine, large intestine, and spleen, and perhaps a delayed clearance of viral infection in the cecum (fig. b to e) , suggesting the important role of mucosal immunity for controlling early infection in sads-cov in mice. it should be noted that mice (c bl/ j mice in this study) may not be the optimal rodent species for sads-cov infection, as wild rats are more commonly seen in chinese pig farms. in addition, other transmission routes may be considered. recently, pdcov has been shown to possibly spread via the respiratory route in addition to fecal-oral transmission ( ) . therefore, it will be interesting to try intranasal route for the inoculation in rats or the other rodent species to mimic sads-cov natural transmission in future studies. more interestingly, we identified dcs to be the precise cell population that supported sads-cov replication (fig. ) . there have been a few reports of immune cell tropism for covs. macrophages are susceptible to mhv infection, representing the largest group of innate immune cells that infiltrate the central nervous system after infection with neurotropic mhv strains ( ) . in addition, based on the fact that sars-cov spike-pseudotyped hiv-based vectors can efficiently transduce human dcs, kobinger ( ) . these previous evidences support our present results, showing that sads-cov can efficiently replicate in dcs. furthermore, this study gives us a novel inspiration that rodents may potentially serve as susceptible hosts for sads-cov in addition to bats and pigs. of note, the species rhinolophus bat ␣-cov hku , including sads-cov, possesses unique s genes closely related to the betacoronavirus (␤-cov), in a manner similar to some globally distributed rodent ␣-covs ( , , ) , implying an unknown evolutionary connection between the bat ␣-cov hku and rodent ␣-covs. under the field conditions of china, direct contact between pigs and flying bats is a low probability; however, rodents (especially rats) are frequently visible in the swine industry, causing great nuisance due to feed loss. it is possible that as bats prey on insects near pig facilities, they leave feces containing hku -like covs that contaminate pig feed, which is then eaten by pig and rodents that subsequently become carriers of sads-cov. rats and mice are increasingly implicated as external vectors for a wide range of different pig pathogens, such as lawsonia intracellularis ( ) . rodents not only spread pathogens but also harm the practitioners of the swine industry, as they are thought to be the major source of leptospirosis in pigs and piggery workers ( ) . future studies on identifying sads-covpositive samples in rodents near pig farms are warranted to test this hypothesis. in addition to rodents, we also measured the sads-cov susceptibility of cell lines from humans, monkeys, chickens, and dogs, revealing a remarkably broad spectrum of tropism (table and fig. ) . as for the ability of sads-cov to grow efficiently in human cell lines, we should not underestimate the risk that this bat-origin cov may "jump" from pigs to humans. it is noteworthy that camel workers with high rates of exposure to camel nasal and oral secretions had evidence of mers-cov infection ( ) . considering that sars-cov and mers-cov originated from bats and spread from one species to another through intermediate hosts (civets and camels, respectively), sads-cov may pose a similar risk to human health through transmission from pigs or other susceptible hosts. the cell susceptibility study and testing of the overexpression of four known cov receptors in nonsusceptible mdck cells (fig. ) demonstrated that sads-cov might use a new receptor molecule that is conserved in bats, pigs, rodents, chickens, monkeys, and humans, indicating a low barrier to cross-species transmission. this is in line with the unusual feature of broad species tropism of sads-cov. in summary, these results provide important insights into the ecology of this bat-origin cov, highlighting the possibility of its ability to jump interspecies barriers and the potential role of rodents as susceptible hosts in the field. identification of the unknown sads-cov cellular receptor and further surveillance of other animal populations are needed to fully understand the biology of sads-cov. the sads-cov isolate ch/gd- / at passage was used in all experiments and cultured in vero cells ( ) . the virus was passaged serially using the culture supernatant to infect fresh vero cells at a multiplicity of infection (moi) of . , and viral titers were determined in vero cells by endpoint dilution as the % tissue culture infective dose (tcid ). rabbit polyclonal antibodies (pab) against the membrane (m), nucleocapsid (n), and the nonstructural protein (nsp ) acidic domain (ac) of sads-cov were generated in-house and validated in sads-cov-infected vero cells ( ) . a mouse anti-sads-cov-n pab was also produced to allow double staining when mixed with the rabbit pab. a monoclonal antibody (mab) against dsrna (anti-dsrna mab j ; catalog number j - , scicons, hungary) was used to specifically detect viral replication of sads-cov. cell lines and cell culture. twenty-four cell lines derived from tissues of different species were used (table ) , including human (huh- , hepg /c a, t, a , and hela), monkey (marc- , cos- , bsc- , and vero), swine (st, pk , llc-pk , and ipec-j [ ] ), bat (bfk [ ] and tb- ), canine (mdck), mouse (nih/ t and raw . ), hamster (bhk- and cho), rat (brl- a and nrk- e), and chicken (df- ) cell lines and a primary kidney cell line from mongolian gerbils (prepared in-house). the bfk cell line was a generous gift from changchun tu at the institute of military veterinary medicine, changchun, china. each cell line was cultured in dulbecco's modified eagle's medium (dmem; hyclone) supplemented with % (vol/vol) fetal bovine serum (fbs; biological industries), u/ml penicillin, and u/ml streptomycin under °c, % co , and water-saturated humidity conditions. to determine viral susceptibility, each cell line was cultured at % confluence in -well plates with maintenance medium (mm) containing dmem, . % tryptose phosphate broth (tpb), and % penicillin-streptomycin or mm with addition of g/ml trypsin (mmt) (catalog number t - tab; sigma, st. louis, mo, usa). after cells were washed with phosphate-buffered saline (pbs), they were inoculated with sads-cov diluted in mm or mmt at an moi of . for h. nonattached viruses were removed by washing the cells three times with dmem, and cell monolayers were subsequently incubated in mm or mmt at °c for days. to determine the effect of trypsin on viral entry, cell monolayers were infected by sads-cov in the following three conditions: (i) no trypsin treatment, infected with sads-cov diluted in mm, subsequently incubated in mm; (ii) pretrypsin treatment, inoculated with sads-cov diluted in mmt, subsequently incubated in mm; and (iii) double-trypsin treatment, inoculated with sads-cov in mmt, subsequently incubated in mmt. supernatants from cells were collected at , , , , , and hours postinfection (hpi) for one-step quantitative rt-pcr analysis. cell cultures were examined for cytopathic effects (cpes) and immunofluorescence assay at to hpi. ifa for cell line susceptibility. different cells infected with sads-cov in -well plates were washed twice with pbs and fixed in % paraformaldehyde in pbs and then permeabilized with . % triton x- in pbs. cells were then incubated with the rabbit anti-sads-cov-m pab at : , dilution for h at °c, washed with pbs, and stained with the alexa fluor -conjugated goat anti-rabbit secondary antibody (thermo fisher scientific, usa) at : , dilution. after incubation for h at °c, the cells were washed with pbs, stained with =, -diamidino- -phenylindole (dapi) at : , dilution and visualized on a fluorescence microscope. one-step quantitative rt-pcr analysis targeting the n gene. the full-length sads-cov n gene was inserted into an appropriately digested pet- a vector using two unique restriction sites, namely, ndei and xhoi, and then linearized with xhoi. the n gene was in vitro transcribed using the t high-efficiency transcription kit (transgen biotech co., ltd., beijing china). standard curves were generated using dilutions of a known quantity of the n gene rna to allow absolute quantitation of sads-cov rna copy numbers in samples. total rna was extracted from culture supernatants or tissue homogenates using trizol (thermofisher scientific, usa) following the manufacturer's instructions. sads-cov rna titer was determined by one-step qrt-pcr (toyobo co., ltd.) targeting the n gene with the primers =-ctaaaactagccccaca ggtc- = and =-tgattgcgagaacgagactg- = and the probe -carboxyfluorescein (fam)-gaaacccaa actgaggtgtagcagg- -carboxytetramethylrhodamine (tamra). samples with a cycle threshold value of Ͻ were considered positive based upon validation data using the rna standards. mouse infections, tissue harvest, and viral load determination. wild-type c bl/ j mice (catalog number ; jackson laboratory) were purchased from the model animal research center of nanjing university and housed in animal facilities at the zhejiang university under specific-pathogen-free conditions. for sads-cov infections, -to -week-old female and male mice were inoculated with ϫ tcid (equal to ϫ genome copies) of sads-cov, either per oral (p.o.) infection with -l inoculum ( ϫ tcid /ml) or intraperitoneal (i.p.) infection with -l inoculum ( . ϫ tcid / ml). for viral load determination in specific tissues, mice were euthanized at , , , , , , and days postinfection (dpi), and tissues were harvested, including stomach, duodenum, jejunum, ileum, cecum, colon, mesenteric lymph nodes, spleen, kidney, liver, heart, lung, blood, and feces. tissues were weighed and homogenized in medium (dmem contained % fbs) by bead beating using sterile zirconium oxide beads (catalog number zrob ; midsci). total rna was extracted from tissue homogenates and tested by quantitative rt-pcr analysis targeting the sads-cov n gene, as described above. blood samples were collected from the heart, and serum was separated for virus-specific antibody detection. enzyme-linked immunosorbent assay. sads-cov virus particles were purified from infected cell culture supernatants by sucrose density gradient centrifugation, and protein concentration was determined by the bicinchoninic acid (bca) protein assay kit (beyotime biotechnology, shanghai, china). the optimal dilution of antigen was determined by square titration. the igg antibodies contained in serum at a : dilution were detected in wells coated with purified sads-cov virus particles ( . ng/well) as antigen. histopathology, immunohistochemistry, and immunofluorescence assay for murine spleen. mice were infected i.p. with sads-cov, and at dpi, spleens were harvested and fixed in % paraformaldehyde for h and embedded in paraffin. tissue sections were then deparaffinized and rehydrated in three changes of xylene, min each, dehydrated in two changes of pure ethanol for min, followed by rehydration in an ethanol gradient of % and % ethanol. after tissues were washed in distilled water, they were subjected to hematoxylin and eosin staining for histopathological examinations. for antigen retrieval, deparaffinized and rehydrated sections were immersed in sodium citrate antigen retrieval solution (ph . ) and maintained at a subboiling temperature for min, were left at °c for min, and then incubated again at subboiling temperature for min. after the sections were allowed to cool to room temperature (rt) and were washed three times with pbs (ph . ), endogenous peroxidase was blocked by immersion in % hydrogen peroxide at rt for min, and sections were again washed with pbs. tissue sections were blocked in % bovine serum albumin (bsa) at rt for min and then incubated with a : dilution of each primary antibody (anti-dsrna mab, anti-sads-cov-m pab, or anti-sads-cov-ac pab) overnight at °c. after slides were washed three times with pbs (ph . ), they were stained with appropriate secondary antibodies labeled with horseradish peroxidase at rt for min. freshly prepared diaminobenzidine chromogenic reagent was added and counterstained with hematoxylin and then dehydrated and visualized on a light microscope. spontaneous fluorescence quenching reagent (wuhan servicebio technology co., ltd., wuhan, china) was added to the tissue sections and incubated for min after antigen retrieval. the sections were then washed in running water, followed with blocking and antibody staining as described above. in addition, the primary antibody was supplement with a cd c antibody (wuhan servicebio technology) at a : dilution and then stained with appropriate secondary antibodies. finally, dapi was added, and sections were visualized on a fluorescence microscope; nuclei labeled with dapi appear blue, and positive cells are green by labeling with cd /c or red by labeling with a virus-specific antibody. preparation of murine splenocytes and flow cytometry. mice infected with sads-cov were euthanized at dpi, and spleens were removed and placed in ml complete dmem. after the excised spleen was ground through a -m cell strainer using the plunger end of a -ml syringe, cells were washed with an excess of dmem and centrifuged at ϫ g for min. after cells were resuspended in ml of red blood cell lysis buffer (solarbio life sciences, beijing, china) and incubated at rt for min, ml of dmem was added, and cells were passed through another strainer to remove clumps. after centrifugation at ϫ g for min, the supernatant was discarded, and cells were resuspended in ml fresh dmem for cell counting and viability checks using trypan blue and a hemocytometer. for flow cytometry, cultured cells were resuspended in fc block buffer (containing anti-mouse cd fc block antibody at : dilution) and incubated on ice. cells in fc block buffer were added to -well plates at ϫ cells/well. after a -min incubation, cells were centrifuged at ϫ g for min, the supernatant was discarded, and pellets were resuspended in a -l cytofix/cytoperm solution (cytofix/cytoperm soln kit; bd biosciences, san jose, ca, usa) to fix cells. after incubation on ice for min protected from light, cells were centrifuged at ϫ g for min at °c, supernatant was removed without disturbing cell pellets, and cells were washed twice in l of ϫ perm/wash buffer. after the addition of -l virus-specific primary antibody (anti-dsrna mab, anti-sads-cov-n pab, or anti-sads-cov-ac pab) diluted in ϫ perm/wash buffer with % bsa and incubation at °c for min, cells were centrifuged at ϫ g for min. cells were washed twice in l of ϫ perm/wash buffer followed by staining with appropriate secondary antibodies conjugated to alexa fluor (thermo fisher scientific, usa) at °c for min. after pellets were centrifuged at ϫ g for min at °c and washed with ϫ perm/wash buffer, they were resuspended in . ml fluorescence-activated cell sorter (facs) buffer and analyzed by flow cytometry. to detect replication of sads-cov in mouse splenic cells in vitro, splenocytes were extracted from naive mice, plated in -or -mm dishes, and infected with sads-cov at an moi of . . at hpi, splenocytes were harvested and placed in a -ml tube, centrifuged at ϫ g for min at °c, and analyzed by flow cytometry as described above. infected mouse splenic cells in -mm dishes were detected by immunofluorescence assay with anti-sads-cov-n antibodies, and infection supernatants were collected at , , , , , and hpi for one-step quantitative rt-pcr analysis. facs analysis of splenocytes with cell marker staining. mice infected with sads-cov were euthanized at dpi, and splenocytes were prepared for flow cytometry by staining with the following appropriate antibodies: anti-sads-cov-ac following secondary antibodies conjugated to alexa fluor (thermo fisher scientific, usa), anti-cd -fitc (catalog number ; ebioscience) for b cells, anti-cd -pe (catalog number ; ebioscience) for t cells, anti-cd /c-pe-cy (catalog number ; bd bioscience) for dcs, and anti-f / -pe/cy (catalog number ; biolegend) for macrophages. stained cells were resuspended in . ml facs buffer and analyzed by flow cytometry. production and transduction of s protein-pseudotyped lentiviruses. pseudovirions with various cov spike proteins were produced as described previously ( ) . briefly, each of the plasmids encoding tgev, sars-cov, mers-cov, and mouse hepatitis virus (mhv) s proteins were cotransfected into t cells with plenti-luc-green fluorescent protein (gfp) and pspax plasmids (kindly provided by zhaohui qian, chinese academy of medical sciences & peking union medical college) at a molar ratio of : : by using polyethylenimine (pei). the cells were fed with fresh medium in the next h, and the supernatant media containing pseudovirions were then collected and centrifuged at ϫ g for min to remove debris. to quantify s protein-mediated entry of pseudovirions, mdck cells were seeded at about % to % confluence in -well plates and transfected with either papn-flag, hdpp -flag, mceacam a-flag, hace -gfp (kindly provided by zhaohui qian) ( ) , or the control backbone vector by using lipofectamine (thermo fisher). the mdck cells overexpressing each receptor were inoculated with l of : diluted corresponding pseudovirions at h posttransfection. at hpi, cells were lysed at room temperature with l of medium with an equal volume of steady-glo (promega, madison, wi). the cell lysates were also used to confirm the expression of each receptor by using western blotting. transduction efficiency was monitored by quantitation of luciferase activity using a modulus ii microplate reader (turner biosystems, sunnyvale, ca). on the other hand, the mdck cells overexpressing each receptor were inoculated with sads-cov (moi, ) at h posttransfection. ifa was performed to test for sads-cov susceptibility using anti-n pab. the replication competency of sads-cov in mdck cells was further determined by a reverse genetics system. development of a dna-launched sads-cov (seacov) infectious cdna clone (named psea) and rescue of sads-cov by transfection of cultured cells with psea followed by passaging on vero cells have been described recently by our lab ( ) . ethics statement. all animal experiments were performed in strict accordance with the experimental animal ethics committee of zhejiang university (approval number zju ). recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans feline coronavirus antibody titer in cerebrospinal fluid from cats with neurological signs origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states bat-to-human: spike features determining "host jump" of coronaviruses sars-cov, mers-cov, and beyond a novel coronavirus associated with severe acute respiratory syndrome ecology, evolution and classification of bat coronaviruses in the aftermath of sars middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease mers, sars and other coronaviruses as causes of pneumonia coronavirus spike protein and tropism changes discovery of a novel swine enteric alphacoronavirus (seacov) in southern china complete genome sequence of bat coronavirus hku from chinese horseshoe bats revealed a much smaller spike gene with a different evolutionary lineage from the rest of the genome a new bat-hku -like coronavirus in swine fatal swine acute diarrhoea syndrome caused by an hku -related coronavirus of bat origin bat-origin coronaviruses expand their host range to pigs propagation of the virus of porcine epidemic diarrhea in cell culture proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture angiotensin-converting enzyme is a functional receptor for the sars coronavirus dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev porcine deltacoronavirus engages the transmissible gastroenteritis virus functional receptor porcine aminopeptidase n for infectious cellular entry broad receptor engagement of an emerging global coronavirus may potentiate its diverse cross-species transmissibility receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins characterization of a novel bat-hku -like swine enteric alphacoronavirus (seacov) infection in cultured cells and development of a seacov infectious clone characterization of a novel g p[ ] rotavirus isolated from a lesser horseshoe bat: a distant relative of feline/canine rotaviruses differential cell line susceptibility to the emerging novel human betacoronavirus c emc/ : implications for disease pathogenesis and clinical manifestation human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines coronavirus hku in respiratory tract of pigs and first discovery of coronavirus quasispecies in =-untranslated region protective role of toll-like receptor -induced type i interferon in murine coronavirus infection of macrophages human immunodeficiency viral vector pseudotyped with the spike envelope of severe acute respiratory syndrome coronavirus transduces human airway epithelial cells and dendritic cells ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign shared common ancestry of rodent alphacoronaviruses sampled globally discovery, diversity and evolution of novel coronaviruses sampled from rodents in china colonisation and shedding of lawsonia intracellularis in experimentally inoculated rodents and in wild rodents on pig farms leptospirosis in piggery workers on trinidad high prevalence of mers-cov infection in camel workers in saudi arabia aminopeptidase-nindependent entry of porcine epidemic diarrhea virus into vero or porcine small intestine epithelial cells identification of the fusion peptide-containing region in betacoronavirus spike glycoproteins the professional editing service nb revisions was used for technical preparation of the text prior to submission. key: cord- -yc q z authors: qian, zhaohui; dominguez, samuel r.; holmes, kathryn v. title: role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: yc q z little is known about the biology of the emerging human group c betacoronavirus, middle east respiratory syndrome coronavirus (mers-cov). because coronavirus spike glycoproteins (s) mediate virus entry, affect viral host range, and elicit neutralizing antibodies, analyzing the functions of mers-cov s protein is a high research priority. mers-cov s on lentivirus pseudovirions mediated entry into a variety of cell types including embryo cells from new world eptesicus fuscus bats. surprisingly, a polyclonal antibody to the s protein of mhv, a group a murine betacoronavirus, cross-reacted in immunoblots with the s domain of group c mers-cov spike protein. mers pseudovirions released from t cells contained only uncleaved s, and pseudovirus entry was blocked by lysosomotropic reagents nh( )cl and bafilomycin and inhibitors of cathepsin l. however, when mers pseudovirions with uncleaved s protein were adsorbed at °c to vero e cells, brief trypsin treatment at neutral ph triggered virus entry at the plasma membrane and syncytia formation. when t cells producing mers pseudotypes co-expressed serine proteases tmprss- or - , large syncytia formed at neutral ph, and the pseudovirions produced were non-infectious and deficient in s protein. these experiments show that if s protein on mers pseudovirions is uncleaved, then viruses enter by endocytosis in a cathepsin l-dependent manner, but if mers-cov s is cleaved, either during virus maturation by serine proteases or on pseudovirions by trypsin in extracellular fluids, then viruses enter at the plasma membrane at neutral ph and cause massive syncytia formation even in cells that express little or no mers-cov receptor. thus, whether mers-cov enters cells within endosomes or at the plasma membrane depends upon the host cell type and tissue, and is determined by the location of host proteases that cleave the viral spike glycoprotein and activate membrane fusion. coronaviruses cause respiratory, enteric, renal and/or neurological disease in humans, many other mammals and birds. in - a previously unknown coronavirus emerged from a wild animal reservoir to cause the sars pandemic, with about , human cases and more than deaths [ , ] . previously, cross-species transmission from coronaviruses of bat and bovine origin had allowed human respiratory coronaviruses oc , nl and e to become established in the human population worldwide [ ] [ ] [ ] [ ] [ ] [ ] . in the arabian peninsula in , another novel human cov, now called middle east respiratory syndrome coronavirus (mers-cov), was isolated in vero e cells from sputum from a fatal case of severe respiratory disease with kidney failure. since then, mers-cov rna has been detected by rt-pcr in over patients with severe to moderate respiratory disease, of whom have died [ , ] . genome sequence analysis showed that mers-cov is a novel betacoronavirus in genogroup c, closely related to two prototype group c betacoronaviruses of asian bats, btcov-hku from a tylonycteris pachypus bat and btcov-hku from a pipistrellus abramus bat [ ] , and to partial sequences of a group c betacoronavirus from a pipistrellus pipistrellus bat in the netherlands [ ] . recently group c betacoronaviruses were also detected in a nyctinomops laticaudatus bat in mexico [ ] , and nycyteris cf. gambiensis bats in ghana [ ] . mers-cov, like sars-cov, is probably a zoonotic betacoronavirus that has spilled over into humans, directly or indirectly, from one of the species of bats that harbor group c betacoronaviruses or from other unknown animal reservoirs [ , , ] . the ~ kda spike glycoprotein (s) of coronaviruses is an important determinant of virus virulence, tissue tropism and host range. trimers of s form the characteristic large spikes on the coronavirus envelope that bind to receptors, mediate membrane fusion, virus entry and syncytia formation, and elicit virus neutralizing antibodies. coronavirus s proteins are class i viral fusion proteins like the hiv envelope (env), influenza hemagglutinin (ha) and paramyxovirus fusion (f) glycoproteins [ ] , which typically require protease cleavage between the s and s domains ( figure a ) to permit conformational changes in s , activated by receptor binding and/or low ph, that mediate membrane fusion leading to virus entry and syncytia formation [ , , ] . in different cell types and tissues, coronavirus s proteins may be cleaved by a variety of host proteases including furin, trypsin, human airway trypsin-like protease (hat), transmembrane protease serine protease- (tmprss- ), tmprss- , or cathepsins [ ] [ ] [ ] [ ] [ ] . functional analysis of mers-cov s glycoprotein is needed to identify susceptible cell types and host species that affect viral tissue tropism and host range, and to determine how various host proteases promote mers-cov virus entry and syncytia formation. identification of the receptor or receptors is an important first step in understanding the host range and tissue tropism of coronaviruses. four receptor proteins for spike proteins of different coronaviruses are now known: murine carcinoembryonic antigen cell adhesion molecule a (mceacam a) for mouse hepatitis virus (mhv) [ ] , a betacoronavirus in group a; aminopeptidase n (apn) for human coronavirus e (hcov- e) and several other alphacoronaviruses [ , ] ; and angiotensin-converting enzyme (ace ) for both sars-cov, a betacoronavirus in group b and hcov-nl , an alphacoronavirus [ , ] . raj and co-workers [ ] recently demonstrated that mers-cov uses dipeptidyl peptidase (dpp ) as a receptor. in contrast, s proteins of several group a betacoronaviruses including bovine coronavirus and hcov-oc use sialic acid moieties as receptors [ , ] . we have used lentivirus pseudotypes with mers-cov spike glycoprotein to identify cells susceptible to infection with mers-cov and to study the role of mers s protein in virus entry and syncytia formation. expression of coronavirus s proteins on t cell membranes for incorporation into lentivirus pseudovirions can be enhanced by using codon-optimized spike cdna and deleting an er/golgi retention motif and an endosomal recycling motif from the cytoplasmic tail of s [ ] [ ] [ ] . codonoptimized cdna encoding s of mers-cov (derived from genbank: afs ) [ ] , with the c-terminal amino acids replaced by a linker, ggggs, and a flag tag (here called mers-cov sΔ ) ( figure a ) was expressed on t cell figure b, lane ) . no protease cleavage products of the ~ kda s protein were detected in transfected t cells or pseudovirions ( figure b) . in marked contrast, the mers lentivirus pseudovirions used to identify cells susceptible to entry of mers-cov in the poehlmann laboratory [ ] , contained a high proportion of cleaved mers-cov s protein at about kda. this important difference in the mers pseudovirions is likely due to differences between our t cells and those used in the poehlmann laboratory. surprisingly, when these mers pseudovirions and cell lysates were blotted with polyclonal goat antibody ao to spikes purified from detergent-disrupted virions of mhv-a , a betacoronavirus in group a, the mers s protein bands were detected ( figure b ). immunoblotting of soluble, truncated mers s proteins with c-terminal flag tags showed that the ao antibody did not recognize the s domain of mers s ( figure c ), so the cross-reactivity between these proteins from betacoronavirus groups a and c must lie within the s domain. vero e and llcmk monkey kidney cell lines are susceptible to infection with mers-cov virus and to sars-cov [ , ] , and also susceptible to sars pseudovirions and to mers pseudovirions with uncleaved s protein ( figure a ). cell entry was quantitated by expression of the luciferase reporter gene in pseudovirus-transduced cells. compared to control pseudovirions with no spike protein, mers pseudovirions showed a to , fold increase in luciferase activity in vero e and llcmk cells (figure a and b), and sars pseudovirions showed a , increase in luciferase activity in vero e cells. because the uncleaved mers-cov s protein mediated virus entry into vero e and llcmk cells, transduction by mers pseudovirions was used to identify additional cell lines that express functional receptors for mers-cov [ , ] . mers pseudovirions detected strong mers-cov receptor activity on the calu line of human airway epithelial cells (figure a and b) , and weaker receptor activity on the a line of human alveolar basal epithelial cells ( figure a ) as also shown by mers-cov infection [ ] . interestingly, the eff embryo cell line from eptesicus fuscus bats was susceptible to mers pseudovirions, increasing luciferase activity by nearly -fold compared to the no spike control, but the tb lu lung cell line from tadarida brasiliensis bats, murine fibroblasts and hela cells were not susceptible to mers pseudovirions ( figure c ). expression of human ace in t cells did not significantly increase susceptibility to mers pseudovirions ( figure a ), although as expected hace greatly increased susceptibility of t cells to sars pseudovirions ( figure a ). figure b and c show that neither human ceacam , or four related human ceacam proteins or human apn functions as a receptor for mers-cov spike protein. these experiments confirm the observation that mers-cov does not use the receptor proteins known for other coronaviruses [ ] or related human membrane proteins. instead dpp is the principal receptor protein for mers-cov [ ] . mers pseudovirions induced a small but consistent to -fold increase in luciferase activity in t human embryo kidney cells compared to the no spike control virus ( figure c ), suggesting that our t cells expressed either a low level of dpp , or an alternative but less efficient receptor, such as cd l or lsectin for sars-cov [ , ] . to determine whether entry of mers pseudovirions with uncleaved s protein required endocytosis and acidification in endosomes, the effects of ammonium chloride and bafilomycin a, lysosomotropic agents that inhibit the acidification of endosomes, were studied. in vero e cells, mm nh cl inhibited entry of sars pseudovirions by about . % compared to entry of sars pseudovirions without inhibitor, and nh cl also inhibited entry of mers pseudovirions by about . % ( figure a ). bafilomycin a specifically inhibits the vacuolar-type h+-atpase that is required for acidification of lysosomes. figure a shows that bafilomycin a inhibited entry of sars pseudovirions into vero e cells by . % as previously reported [ , ] , and also inhibited entry of mers pseudovirions by more than . % compared to mers pseudovirions without inhibitor. in llcmk cells, although bafilomycin a inhibited . % of mers-cov s mediated entry, nh cl reduced mers-cov s-mediated entry only -fold (data not shown), suggesting that the inhibition of endosomal acidification by nh cl may be cell type dependent. these experiments show that mers pseudovirions with uncleaved s protein can enter monkey kidney cells only by endocytosis. cathepsins are a diverse group of acid-activated cysteine proteases located within endosomes and lysosomes. cathepsin activity is essential for infection by several viruses that enter by the endosomal route, including reovirus [ ] , sars-cov [ ] , and ebolavirus [ ] . e d, an inhibitor of the cysteine protease activities of cathepsins b, h, and l and calpain, reduced transduction of vero e cells by sars pseudovirions by % as previously reported ( figure b ) [ ] . since cell entry mediated by vsv-g glycoprotein does not require protease activation [ ] , e d treatment of vero e ( figure b ) and llcmk cells (data not shown) did not inhibit entry of vsv pseudovirions. however, e d decreased entry into vero e cells of mers pseudovirions with uncleaved s by . % ( figure b ) and llcmk cells by . % (data not shown). thus, cleavage of mers-cov s protein by one of the cathepsins or calpain was required for triggering s-mediated membrane fusion and virus entry at low ph in endosomes. as previously reported [ ] , in vero e cells µm of cathepsin l inhibitor iii, a specific and irreversible inhibitor of cathepsin l, significantly inhibited entry mediated by sars s protein, but did not inhibit vsv-g-mediated entry ( figure b ). cathepsin l inhibitor iii reduced entry into vero e cells of mers pseudovirions with uncleaved s protein by % relative to entry without inhibitor ( figure b ), and similar results were seen in llcmk cells (data not shown). thus, mers-cov s protein on pseudovirions must be cleaved in endosomes by the acidactivated cysteine protease activity of cathepsin l to trigger receptor-dependent entry into vero e and llcmk cells. trypsin cleavage of mers-cov s on pseudovirions adsorbed to receptors on the cell surface triggers virus entry at the plasma membrane at neutral ph sars-cov can enter susceptible cells at the plasma membrane, instead of by endocytosis, if virions adsorbed at °c to ace on the cell membrane are treated with trypsin, then warmed to °c in the presence of an inhibitor of endosomal acidification [ ] . trypsin treatment at either °c or °c cleaved the s protein of mers pseudovirions and generated a ~ kda subunit in the s domain of the protein recognized by antibody to mhv-a s protein ( figure s ). mers pseudovirions with uncleaved s protein were adsorbed at °c to cell surface receptors on vero e cells in the presence of mm nh cl, and then the cells with bound virions were briefly treated with trypsin at ph . at room temperature to cleave the ~ kda s protein and activate its membrane fusing activity. figures a and a show that nh cl strongly inhibited infection of vero e cells by mers pseudovirions with uncleaved s. however, trypsin treatment of the mers pseudovirions bound at neutral ph and °c to the vero e cell membrane triggered both virus entry at the plasma membrane and formation of small syncytia by hours post inoculation ( figure a and b). thus, receptor binding together with protease cleavage and activation of s at neutral ph was sufficient to trigger entry of mers pseudovirions and syncytia formation. in this experiment membrane fusion did not depend upon synthesis of s protein, but syncytia formation was mediated by the cleaved s protein on pseudovirions adsorbed to virus receptor on the cell membrane. although acidic ph is required to activate the cathepsin l activity that allows mers pseudovirions to enter at endosomes, low ph is not required for the conformational changes in trypsin-cleaved mers-cov s protein that mediate entry at the plasma membrane. t cells expressing uncleaved mers-cov s protein or control cells stably transfected with the empty pcdna . vector were overlaid on monolayers of vero e cells in the presence or absence of tpck trypsin ( figure c ). no syncytia formation was induced by t cells with empty vector or t cells expressing mers-cov sΔ without trypsin ( figure c ), but addition of tpck trypsin to the medium triggered formation of massive syncytia in the vero e cells co-cultured for hr with mers-cov s-expressing t cells ( figure c , arrows). large syncytia were also formed after even a brief minute trypsin pre-treatment at ph . and °c of t cells expressing mers-cov s protein, followed by incubation with a -fold excess of soybean trypsin inhibitor before layering the cells over confluent monolayers of vero e cells and incubating at °c for hours ( figure c, lower central panel, arrows) . thus, trypsin cleavage at neutral ph of mers-cov s protein on t cells triggered syncytia formation when s was bound to receptors on susceptible vero e cells. type ii transmembrane serine proteases, including tmprss- and tmprss- , which like trypsin are expressed in the respiratory tract, play important roles in triggering entry of influenza a virus, human metapneumovirus and sars betacoronavirus in group b [ , , [ ] [ ] [ ] [ ] . we therefore transfected t cells with plasmids encoding tmprss- or - , mers-cov sΔ protein, pspax and plenti-gfp-luc and investigated whether s proteins on pseudovirions produced in these cells were cleaved and whether they could infect vero e cells in the presence of nh cl. surprisingly, the pseudovirionproducing t cells expressing either tmprss- or - , formed large syncytia by hrs after transfection ( figure a ), but the mers pseudovirions produced by these cells could not transduce vero e cells in the presence or absence of nh cl ( figure b ). in contrast, without tmprss- or - , the t cells expressing uncleaved mers-cov sΔ did not form syncytia, and pseudovirions that they produced efficiently infected vero e cells, but virus entry was inhibited by nh cl ( figure b ). immunoblots with antibody ao to mhv s or anti-flag (data not shown) revealed that the mers pseudovirions produced in t cells expressing tmprss- or - contained little or no immunoreactive s protein or fragments of s. although the novel group c betacoronavirus mers-cov is highly virulent in humans and can infect cells from several different species, including humans, monkeys, pigs, and some species of bats [ , , ] , little is known about the biology of this virus. because the spike glycoprotein is essential for coronavirus entry, elucidating the functions of the mers-cov spike can provide valuable insight into the pathogenesis of mers-cov, and suggest potential therapeutic interventions. here we used lentivirus pseudovirions with mers-cov spike protein to study s-mediated cell entry at biosafety level . we found that mers pseudovirions, like infectious mers-cov virions [ , ] , readily infected the vero e and llcmk lines of monkey kidney cells, several human respiratory epithelial cell lines, and embryo cells from eptesicus fuscus bats. others have also recently demonstrated that human respiratory tract cells, and also primary human bronchus and alveolar cells are susceptible to mers-cov in accord with the severe respiratory disease in mers patients [ , , , ] . muller et al. [ ] recently reported that mers-cov can infect cells from four genera of old world bats, rousettus, rhinolophus, pipistrellus, and myotis, and one new world genus, carollia. we found that mers pseudovirions could also infect cells from one new world bat, e. fuscus, but not from another, t. brasiliensis. the ability of mers-cov to infect cells from multiple mammalian species directly and without adaptation [ ] , including a diverse array of both old world and new world bats, suggests that the receptor for mers-cov, dpp [ ] , is broadly conserved among many species, an important property of many emerging viruses [ , ] . e. fuscus figure a were inoculated onto vero e cells in the presence (striped bars) or absence (white bars) of mm nh cl to inhibit acidification of endosomes. doi: . /journal.pone. .g bats, commonly known as big brown bats, are the bats most commonly encountered by humans in north america, and they are a reservoir for an alphacoronavirus [ , ] . it will be important to learn whether these new world bats are susceptible to mers-cov or related group c betacoronaviruses. the recent detection in n. laticaudatus bats in mexico of a group c betacoronavirus with % similarity to mers-cov [ ] , coupled with the diverse array of alphacoronaviruses previously discovered in north american bats [ ] [ ] [ ] [ ] , justify increased surveillance to identify additional species of new world bats that may also harbor group c betacoronaviruses like mers-cov or other coronaviruses with the potential to cause severe disease in humans. mers-cov is a betacoronavirus in group c, and we were surprised that its s protein was recognized in immunoblots by a polyclonal antibody to the spike protein of mhv-a , a group a betacoronavirus. the cross-reactive epitope(s) was mapped to the s domain, which is more highly conserved than the s domain of betacoronaviruses. chan et al. [ , ] found that mers-cov s protein was recognized in immunofluorescence and in vitro neutralization assays by sera of some convalescent sars patients, and suggested, based on bioinformatics, that epitope(s) in s could account for the observed serological cross-reactivity. these observations that the s protein of mers-cov, a group c betacoronavirus, contains crossreacting epitope(s) with s proteins of both some group b (sars-cov) and group a (mhv) betacoronaviruses, indicate that serological studies may not accurately distinguish between different phylogenetic groups of betacoronaviruses. identification and characterization of the cross-reacting epitope(s) is an important research priority to show whether there is a common epitope in s that could be used as an immunogen to vaccinate against all betacoronaviruses. enveloped viruses infect cells by fusion of the viral envelope with host cell membranes, a process mediated by a series of conformational changes in the viral fusion protein that are regulated by receptor binding, protease activation, and/or ph [ ] . the classes of viral fusion proteins are determined based on their structures and conformational changes during membrane fusion. most class i viral fusion proteins require proteolytic cleavage upstream of the hydrophobic fusion peptide in the viral spike protein to enable these conformational changes to occur, as well as subsequent steps that trigger membrane fusion including either binding to receptor like hiv gp , low ph in endosomes like influenza ha, or both like avian sarcoma leukosis virus (aslv) [ , , [ ] [ ] [ ] . coronaviruses in different phylogenetic groups differ in the sequence of steps leading to virus entry [ , ] . the s protein on virions of group a betacoronavirus mhv-a requires protease activation--either by furin during virus maturation [ ] , by trypsin or other serine proteases in extracellular fluids before either receptor binding, or by cathepsin in endosomes at acidic ph-to trigger the conformational changes that lead to membrane fusion and virus entry [ ] . in contrast, virions of group b betacoronavirus sars-cov that contain uncleaved s [ ] , first bind to the viral receptor protein, ace , and are endocytosed, and then s is cleaved within endosomal vesicles by acid-dependent cathepsin l enabling the conformational changes in s that lead to virus entry [ , , ] . here we analyzed the steps needed to trigger conformational changes in mers-cov s and their roles in virus entry and syncytia formation. in our laboratory, mers pseudovirions released by t cells contained only uncleaved s protein, and, as for most coronaviruses, cleavage between s and s was necessary to enable its membrane fusing activity. the mers pseudovirions bound to receptors on susceptible cells and were endocytized, and within the endosomes cleavage of s by the acid-dependent cysteine protease cathepsin l mediated virus entry. gierer et al [ ] reached similar conclusions using mers pseudovirions that, unlike ours, contained more cleaved than uncleaved s protein, although both labs had made the pseudovirions in t cells. gierer et al. [ ] showed that batches of t cells differ markedly in expression of the mers-cov receptor and susceptibility to transduction by mers pseudovirions. in our laboratory, the t cells showed minimal susceptibility to transduction with mers-cov pseudovirions with uncleaved s protein. in addition to entry by endocytosis, we showed that, like sars-cov [ , ] , mers pseudovirions could enter susceptible vero e cells at the plasma membrane if virions were first bound to cell surface receptors at °c at neutral ph in the presence of nh cl to inhibit acidification of endosomes, and also treated briefly at room temperature with trypsin to cleave the viral s protein. upon warming to °c at neutral ph, the mers pseudovirions fused with the plasma membrane and transduced the cells. thus, mers s protein does not require acidification to mediate virus entry, and the acidification required for endosomal entry [ ] was required to activate the protease activity of cathepsin. although treatment of coronavirus virions or pseudovirions with proteases can activate virus entry, it may also make them lose infectivity if the cleaved s /s heterodimer dissociates before receptor binding. we found that mers pseudovirions released from cells expressing tmprss- contained reduced amounts of s protein and had lost the ability to transduce susceptible cells. we postulate that the mers pseudovirions that contain large amounts of cleaved s protein, detected by a c-terminal tag [ , ] , could not enter cells at the plasma membrane because s may have dissociated from the cleaved spikes on virions. development of antibodies specific for the s domain of mers s protein are needed to test this hypothesis. coronavirus s proteins expressed on cell membranes can trigger receptor-dependent syncytia formation if the membranebound s protein is cleaved within the infected cells by furin or other proteases. mers-cov infection of calu- and caco- cell lines induced syncytia formation [ ] . our t cells were only minimally susceptible to entry of mers-pseudovirions, and did not form syncytia when producing mers pseudovirions with uncleaved s. however, when t cells expressing mers-cov sΔ protein were co-cultured in the presence of trypsin with vero e cells that express the mers-cov receptor, enormous syncytia formed. these observations suggest that in tissues such as the lung, where trypsin, tmprss- or - and hat and other serine proteases are available, mers-cov virus infection might spread directly from cell to cell by s-mediated, receptor-dependent syncytia formation, potentially escaping from virus-neutralizing antibodies, as do other syncytia-forming viruses such as respiratory syncytial virus, parainfluenza viruses, and measles. it will be important to learn whether syncytia are formed in lungs or other tissues of mers-cov patients or animal models of mers-cov. some coronavirus s proteins can also trigger receptorindependent syncytia formation [ , ] . when s proteins expressed on the plasma membrane are cleaved, the s domain can detach from the spike, exposing the hydrophobic fusion peptide of the membrane-anchored s domain that can directly induce fusion with any nearby cell membranes or lipid bilayers even if they lack receptors. this "receptor-independent spread (ris)" allows an infected cell to fuse with adjacent noninfected, receptor-negative cells that can, in turn, produce virus and fuse with additional receptor-negative cells. ris activity depends on the stability of s /s interactions, and low stability of s /s heterodimers correlates with rapid spread of infection through tissues that express little receptor protein [ , ] . we found that transmembrane serine proteases tmprss- and - could activate the syncytia forming activity of mers-cov sΔ protein expressed in t cells as these proteases do for class fusion proteins of other respiratory viruses including influenza, sars-cov and human metapneumovirus [ , , [ ] [ ] [ ] [ ] . we were surprised that t cells, which express very little mers-cov receptor, were so extensively fused, and we hypothesize that this syncytia formation may be due to ris. due to its high case fatality rate, therapeutic interventions for mers-cov are urgently needed. pooled purified human immunoglobulin containing neutralizing antibody has been used to treat a variety of infectious diseases. not surprisingly, since mers-cov is an emerging pathogen, we found that human immunoglobulin from the usa could not neutralize the infectivity of mers pseudovirions (data not shown). however, sera from patients infected with either sars-cov or mers-cov contain antibodies that can neutralize mers-cov [ , ] . most neutralizing antibodies would likely target the receptorbinding s domain of mers-cov s, which is less conserved than the s domain and can mutate, likely generating antibody escape mutants [ ] . here we showed that a polyclonal antibody to the s protein of mhv, a group a betacoronavirus, cross reacts with the s domain of mers s protein in immunoblots. as we and others have proposed for sars-covs [ , ] , the more highly conserved s domain, and especially its c-terminal heptad repeat (hrc) region, can be important targets for blocking conformational changes in s, inhibiting syncytia formation and virus entry, and also eliciting neutralizing antibodies. if mers-cov virions made in the lung have uncleaved s protein and must therefore enter cells through endocytosis, then inhibitory hrc peptides, or neutralizing antibodies to hrc might not penetrate into the endosomes to prevent virus entry. however, mers-cov virions in the lung likely have cleaved s protein due to lung proteases, so that virus entry at the plasma membrane might be inhibited by hrc-targeted peptides or antibodies. in summary, we demonstrated that, similar to sars-cov, cleavage of mers-cov-s protein by trypsin, tmprss or - or cathepsin l is required to activate the membrane fusion activity of s, leading to virus entry and syncytia formation, and that the location of the protease determines whether virus enters via endocytosis or by fusion at the plasma membrane. mers-cov s-mediated binding and entry mechanisms and protease triggering of conformational changes required for mers-cov-s virus entry and syncytia formation present potential targets for development of drugs or vaccines against this newly emerging and lethal group c human betacoronavirus. codon-optimized cdna encoding the spike glycoprotein of mers-cov [ ] was synthesized with the c-terminal amino acids replaced with a ggggs linker and a flag tag (genscript, piscataway, nj), and for eukaryotic expression was cloned into pcdna . (+) (invitrogen) between the bamhi and noti sites. to make constructs for expression of truncated soluble mers s aa - , s aa - , and s aa - proteins, pcr reactions were performed using the same forward primer aatgaaaagcttcaccatgattcactccgtgttcctc pairing with the following reverse primers, for s aa - : tagttttctagaacttccgcctccaccataa ctacagtggagctggct; for s aa - : tagttttctagaacttccgcctccac cgtcgcactccacgccttctgcc; or for s aa - tagttttctagaacttc cgcctccacctggggtcagtgtgctgggggt, and cloned into p xflag-cmv (sigma, st louis, mo) between hindiii and xbai sites for expression. the vsv-g plasmid and lentiviral packaging plasmid, pspax , were obtained from addgene (cambridge, ma). the lentiviral reporter plasmid, plenti-gfp-luc, which expresses green fluorescent protein (gfp) and luciferase, was kindly provided by fang li, duke university [ ] the vero e line of african green monkey kidney cells, the t line of human embryonic kidney cells transformed with sv large t antigen, the calu line of human airway epithelial cells, the a line of human alveolar epithelial cells, and the tb lu lung cell line from t. brasiliensis bats were obtained from atcc (manassas, va). hela cells stably expressing recombinant human ceacam proteins and the control hela cell line containing the empty vector were kindly provided by scott gray-owen, university of toronto [ ] . the bat eff cells were prepared by macerating mid-gestation fetuses of eptesicus fuscus bats, briefly trypsinizing the cells, and plating them for expansion. cells were passaged twice, frozen, and kindly provided by richard bowen, colorado state university. isolation of the eff cells was conducted under approval - a from the colorado state university iacuc. murine nih t cells stably expressing recombinant human aminopeptidase n (hapn) or control cells with empty vector were previously described [ ] . these cell lines were maintained in dulbecco's mem with % fetal bovine serum (fbs) and % penicillin, streptomycin, and fugizone (psf) (life technologies inc, grand island, ny). the llc-mk line of rhesus monkey kidney cells from atcc ccl- was maintained in opti-mem (life technologies inc, grand island, ny) with % fbs and % psf. pseudotyped lentiviruses were produced as described previously [ ] with minor modifications. briefly, plasmids that encode viral spike glycoproteins mers-cov s∆ , sars s∆ , or vsv-g were co-transfected into t cells with pspax and plenti-gfp-luc using polyetherimide (pei) (polyscience inc, warrington, pa). forty to hr later the supernatant media containing pseudovirions were centrifuged at g for min to remove debris, and passed through a . µm filter. to quantitate entry of pseudovirions into different cell types, µl of pseudovirions with µg/ml of polybrene (sigma) was inoculated onto cells in -well plates, incubated overnight at °c, and cells were fed with fresh medium. at hr post inoculation (pi) cells were lysed at room temperature with µl of medium with an equal volume of steady-glo (promega, madison, wi). transduction efficiency was monitored by quantitation of luciferase activity. living cells transduced by pseudovirions were detected by gfp expression. for all experiments triplicate samples were analyzed, data are representative of two or more experiments, and the standard error is shown. pseudovirions with mers-cov sΔ , sars sΔ , or vsvg glycoprotein or control pseudovirions with no spike were centrifuged through a % sucrose cushion at , rpm at °c for h in a beckman sw rotor [ ] . spike proteins in the virions were separated on - % sds page, blotted to nitrocellulose and detected with mouse anti-flag antibody m (sigma, st louis, mo) for mers-cov sΔ , or polyclonal goat antibody ao to purified spikes from detergent-disrupted mhv- virions [ ] , followed by horseradish peroxidase (hrp)-conjugated antibody to mouse or goat igg, and visualized with chemiluminescent reagent plus (perkinelmer, boston, ma). vero e cells or llcmk cells were incubated for hr at °c with medium alone or medium containing either mm nh cl or nm bafilomycin a to inhibit acidification of endosomes, and then spin-inoculated with pseudovirions and no spike controls in the presence of either mm nh cl or nm bafilomycin a for min at , g at °c. at hr pi cells were lysed and luciferase activity was quantitated as a measure of virus entry. in figure , the luciferase activities of vero e cells transduced with vsv-, sars-, and mers-covspseudovirions without endocytosis inhibitor were x , . - x , and - x , respectively. monolayers of vero e or llcmk cells were incubated with mm nh cl in medium containing % fbs for hr at °c, then shifted for min to °c with mm nh cl. pseudovirions were adsorbed to cells at °c by spin-inoculation for min at , g. the virus inocula were removed and replaced with prewarmed, serum-free dmem with or without mm nh cl. after min at °c, the media were replaced with serum-free dmem with or without µg/ml of tpck-trypsin at room temperature to activate the membrane fusing activity of the s protein on virions adsorbed to the plasma membrane. trypsin activity was then inhibited by incubation for min on ice with µg/ml of soybean trypsin inhibitor (worthington biochemical corporation, lakewood, nj) in dmem with % fbs, and cells were incubated overnight at °c, fed with fresh medium, and incubated at °c. at hr pi, virus entry was quantitated by luciferase activity and cells were photographed to detect viral cytopathic effects. monolayers of vero e cells or llcmk cells were preincubated for hrs at °c with either µm of e d, that inhibits cathepsin b, h, l and calpain, or µm of specific cathepsin l inhibitor iii (millipore, billerica, ma). pseudovirions and no spike controls with or without cathepsin inhibitors were spin-inoculated onto the cells at °c, then incubated at °c for hours with or without the endocytosis inhibitors. cells were then incubated at °c without inhibitors, and at hr pi, luciferase activity in lysed cells was determined. t cells in -well plates were transfected using pei with µg of either empty vector or mers-cov s∆ plasmid. for brief trypsin pre-treatment, cells were lifted with mm edta in pbs and washed, then incubated on ice with µg/ml of tpck trypsin or control medium for min. typsin activity was then inhibited by incubation with a five-fold excess of soybean trypsin inhibitor for min. the trypsin-pretreated t cells and control cells were then layered over monolayers of vero e cells and incubated for hr. syncytia formation was detected by phase contrast microscopy or by imaging fixed cells stained with crystal violet. figure s . detection of trypsin-cleaved mers-cov s protein on pseudovirions by antibody to mhv s protein. pseudovirions containing either mers-cov s protein or no spike (control) were incubated with µg/ml of trypsin either at °c or °c for min. after digestion, glycoproteins on pseudovirions were analyzed by immunoblot using ao antibody to the s protein of murine betacoronavirus mhv-a that cross-reacts with mers-cov s protein. lanes to : no spike control; lanes to : mers pseudovirions; lanes and : no trypsin; lanes and : trypsin at °c; lane and : trypsin at °c. the band at ~ kda was seen in pseudovirions without spike, indicating that it is not due to mers-cov s protein. uncleaved mers-cov s protein, kda, on pseudovirions is shown in lane , and trypsin treatment cleaved all of the s protein, and generated a subunit of ~ kda that was recognized by ao antibody (lanes and ). 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in the spike glycoprotein of murine coronavirus are induced at degrees c either by soluble murine ceacam receptors or by ph proteasemediated entry via the endosome of human coronavirus e cell receptorindependent infection by a neurotropic murine coronavirus entry of mouse hepatitis virus into cells by endosomal and nonendosomal pathways the spike glycoprotein of murine coronavirus mhv-jhm mediates receptor-independent infection and spread in the central nervous systems of ceacam a-/-mice enhanced virulence mediated by the murine coronavirus, mouse hepatitis virus strain jhm, is associated with a glycine at residue of the spike glycoprotein escape from human monoclonal antibody neutralization affects in vitro and in vivo fitness of severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus (sars-cov) infection inhibition using spike protein heptad repeat-derived peptides requirements for prediction of peptide retention time in reversed-phase high-performance liquid chromatography: hydrophilicity/hydrophobicity of side-chains at the n-and c-termini of peptides are dramatically affected by the end-groups and location apoptotic caspases regulate induction of ipscs from human fibroblasts cd carcinoembryonic antigens mediate interactions between opaexpressing neisseria gonorrhoeae and human polymorphonuclear phagocytes human myeloid plasma membrane glycoprotein cd (gp ) is identical to aminopeptidase n complementation of a binding-defective retrovirus by a host cell receptor mutant isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid we thank scott d. gray-owen (university of toronto) for providing hela cells stably expressing human ceacam proteins, linda shapiro (university of connecticut) for mouse cells producing human apn, richard bowen (colorado state university) for the bat cell lines, stefan poehlmann (hannover medical school and german primate center) for pca -tmprss and pcmv-tmprss plasmids, and fang li (duke university) for plenti-gfp-luc. conceived and designed the experiments: zq srd kvh. performed the experiments: zq. analyzed the data: zq srd kvh. contributed reagents/materials/analysis tools: zq srd kvh. wrote the manuscript: zq srd kvh. key: cord- -hsc x j authors: dittmar, mark; lee, jae seung; whig, kanupriya; segrist, elisha; li, minghua; jurado, kellie; samby, kirandeep; ramage, holly; schultz, david; cherry, sara title: drug repurposing screens reveal fda approved drugs active against sars-cov- date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: hsc x j there are an urgent need for antivirals to treat the newly emerged sars-cov- . to identify new candidates we screened a repurposing library of ~ , drugs. screening in vero cells found few antivirals, while screening in human huh . cells validated diverse antiviral drugs. extending our studies to lung epithelial cells, we found that there are major differences in drug sensitivity and entry pathways used by sars-cov- in these cells. entry in lung epithelial calu- cells is ph-independent and requires tmprss , while entry in vero and huh . cells requires low ph and triggering by acid-dependent endosomal proteases. moreover, we found drugs are antiviral in lung cells, of which have been tested in humans, and are fda approved including cyclosporine which we found is targeting cyclophilin rather than calcineurin for its antiviral activity. these antivirals reveal essential host targets and have the potential for rapid clinical implementation. coronaviruses represent a large group of medically relevant viruses were historically associated with the common cold. however, in recent years, members of the coronavirus family have emerged from animal reservoirs into humans and have caused novel diseases ( ). first, severe acute respiratory syndrome (sars-cov) emerged in china in , followed by middle east respiratory syndrome (mers-cov) in ( , ) . while sars was, in the end eradicated, mers continues to cause infections in the middle east. beginning in december , into continuing into january , it became clear that a new respiratory virus was spreading in wuhan, china. rapid sequencing efforts revealed a coronavirus closely related to sars, and was named sars-cov- ( ). unfortunately, this virus is highly infectious and has spread rapidly around the world. identification of broadly acting sars-cov- antivirals is essential to clinically address sars-cov- infections. a potential route to candidate antivirals is through the deployment of drugs that show activity against related viruses. previous studies found that the antiviral drug remdesivir, which was developed against the rna-dependent rna polymerase of ebola virus, was also active against sars-cov- in vitro, with promising results in clinical trials ( ) ( ) ( ) . chloroquine, and its derivatives including hydroxychloroquine are approved for use in malaria, and many in vitro studies have found that these drugs are also active against coronaviruses, including sars-cov- ( , ) . this led to early adoption of these agents to treat covid- (the disease caused by sars-cov- infection); however, little efficacy of these agents has been demonstrated in subsequent clinical trials ( ) . it remains unclear why these agents have not been more active in humans. there are currently more than fda approved drugs, and many others that have been tested in humans. we created an in-house library of drugs including ~ fda approved drugs and ~ drug-like molecules against defined molecular targets with validated pharmacological activity. in addition, we purchased drugs with reported anti-sars-cov- activity (e.g. remdesivir). viruses encode unique proteins essential for infection, and most approved antivirals target these virally encoded essential targets. this class of antivirals has been termed direct-acting antivirals. viruses are also dependent on host cellular machineries for successful infection, and drugs that block these activities are host-targeted antivirals. given our dearth of effective treatments, we developed a screening platform that would allow us to identify both direct-acting and host-targeted antivirals that can be potentially repurposed for use against sars-cov- ( ). we developed a specific and sensitive assay to quantify viral infection using a cell-based highcontent approach. we began our studies in african green monkey (cercopithecus aethiops) kidney epithelial cells (vero) because they are routinely used to propagate sars-cov- . they are robustly infected, and thus vero cells are widely used as a model system to screen for antivirals ( , ( ) ( ) ( ) . we screened our in-house repurposing library, identifying only six drugs that were antiviral with low toxicity in the primary screen. given how few candidates emerged, we reasoned that human cells might be a better model of infection and thus tested a panel of human cell lines to identify cells that are easy to grow, and permissive to infection. we found that the human hepatocyte cell line huh . was readily infected with sars-cov- . screening in this human cell line we validated drugs that were active in dose-response experiments and showed a favorable selective index versus toxicity ( ) . these candidates targeted a wide variety of cellular activities, but few were active in vero cells. however, one class, the chloroquines and their derivatives were active in both cell types. the entry pathway of sars-cov- has only begun to be elucidated with much of what we know being inferred from studies of the related sars-cov- ( , ) . the coronavirus glycoprotein, or spike, requires proteolytic processing for entry ( , , ) . this processing can occur outside the cell, or within the endolysosomal compartment ( , ) . both sars-cov- and - engage angiotensin-converting enzyme (ace ) as their plasma membrane receptor ( ) ( ) ( ) . upon binding, the viruses along with the receptor are endocytosed into the cell into a low ph endosomal compartment where there are proteases, including cathepsins, that can cleave spike and allow for entry into the cytosol ( ) ( ) ( ) . since cathepsins require a low ph for activity, chloroquine and its derivatives that neutralize this low ph can effectively block viral entry ( ) ( ) ( ) . recent studies have also identified a plasma membrane-associated serine protease, tmprss , is active against spike, cleaving the protein extracellularly thereby bypassing the requirement for endosomal proteases ( ) ( ) ( ) . whether sars-cov- enters through different routes in different cell types remains unclear. lung epithelial cells are the major cellular target for sars-cov- in vivo and have been used to explore the role of tmprss in infection. perhaps surprisingly, while we found remdesivir was antiviral in calu- , hydroxychloroquine was not. since a panel of quinolines had no activity in calu- cells, these data suggest that entry in lung epithelial cells is independent of low-ph processing in the endosomal compartment. in contrast, the tmprss inhibitor camostat was highly active in calu- cells but inactive in vero and huh . cells. these data demonstrate distinct modes of entry in lung cells ( ) . further, these data suggest that there may be other fundamentally different cellular requirements in different cell types. we screened our validated candidates in calu- cells and found only drugs showed activity, including fda approved drugs: cyclosporine, dacomitinib and salinomycin. in additional studies, we found that cyclosporine analogs that target cyclophilin a were active against sars-cov- , but not compounds that target calcineurin. identifying broadly acting antivirals is essential to move forward with clinical treatments for sars-cov- . vero cells are permissive to infection and can be used for antiviral screening for direct acting antivirals sars-cov- is routinely propagated in vero e cells ( , , ) . when growing the virus in either vero e or vero ccl cells, two different strains of vero cells from atcc, we observed that sars-cov- is cytopathic in vero e , but not in vero ccl (data not shown) ( ) . moreover, viral stocks propagated from either of these cells produced similar titers of virus ( x pfu/ml) suggesting that viral replication and cytotoxicity are separable. therefore, we set out to develop a quantitative microscopy-based assay to measure the level of replication of sars-cov- more directly in infected cells, and we chose vero ccl to uncouple toxicity from infection. we first validated that our antibodies could detect infection of sars-cov- . we used an antibody to dsrna, and to sars-cov- spike (figure a ) ( ) ( ) . we created an in-house library of drugs purchased from selleckchem. this library contains ~ fda approved drugs and ~ drug-like molecules against defined molecular targets with validated pharmacological activity. the library contains known kinase inhibitors, annotated cancer therapeutics, epigenetic regulators, anti-viral/infectives, gpcr and ion channel regulators. the remaining compounds falling into diverse target classes. we next optimized the dose and timing of infection by performing dose-response studies with known antivirals. indeed, we found that hydroxychloroquine and remdesivir were active in vero cells and presented with little cytotoxicity at the active doses ( figure b) ( ) . next, we validated the assay metrics, and observed a z'= . ( figure s ) ( ) . we used this assay pipeline to screen our in-house repurposing library in well plates at a final concentration of µm ( figure c ) ( ) . we quantified the percentage of infected cells as well as the total cell number per well, to allow for exclusion of toxic compounds. we robustly identified the positive control remdesivir as antiviral ( figure s ) ( ) . using a threshold of < % infection and > % viability, as compared to the vehicle control, we identified only six drugs that were antiviral in our primary screen (table s ). this included the natural product nanchangmycin, which we previously found in a drug repurposing screen against zika virus ( ) . nanchangmycin was broadly antiviral against viruses that enter cells through endocytosis, consistent with the role of endosomal acidification for sars-cov- entry in these cells ( ) . we then repurchased powders and validated four of these candidates in a dose-response assays where we observed antiviral activity in the absence of toxicity (figure d ). since vero cells are derived from african green monkeys, we set out to identify a human cell line permissive to infection. to this end, we infected a panel of human cell lines with sars-cov- and monitored infection by microscopy. we initially tested a , calu- , huh , huh . , hepg , hacat, imr , nci-h , cfbe o, and u os cells. we detected less than % infection of a , calu- , huh , hepg , hacat, imr , nci-h , cfbe o, and u os cells (data not shown). interestingly, while huh were largely non-permissive, the derivative cell line huh . cells was permissive to sars-cov- (fig a) . huh . cells are defective in innate immune signaling (rig-i) and are known to be more permissive to many viruses, including hepatitis c virus ( ) . remdesivir and hydroxylchloroquine were antiviral against sars-cov- in huh . cells with ic s that were greater than -fold lower than those observed in vero cells (fig b) . we also found that nanchangmycin was antiviral against sars-cov- in huh . cells (fig s ) .these observations suggest that huh . cells may be more sensitive to some classes of inhibitors, and may reveal antivirals that are selectively active against human targets. we optimized our image-based assay in huh . cells using remdesivir and observed a z'= . ( fig s ) ( ) . we screened our repurposing library at nm quantifying both the percentage of infected cells as well as cell number to exclude toxic compounds (fig c) . we found drugs had antiviral activity in the absence of cytotoxicity (< % infection, > % viability, as compared to vehicle control) (table s ). this included three of the six drugs identified in vero cells: z-fa-fmk, y- and salinomycin. we repurchased powders for drugs and tested their activity in dose-response assays in huh . cells against sars-cov- . cell number and the percent of infected cells were quantified. remdesivir and hydroxychloroquine were used as positive controls and vehicle controls (dmso) was included as a negative control ( ) . of those tested, drugs showed activity and fell into diverse classes (fig d) . dose-response curves are shown for candidates and the ic s and cc s were calculated (fig e) . the selectivity index (si, ratio between antiviral and cytotoxicity potencies) was calculated and the candidates were antiviral with si> ( figure e , table s ). dose-responses curves for the other candidates that did not validate in huh . cells are shown in fig s . direct-acting antivirals are likely to be active against the virus in multiple cell types, as was observed for remdesivir. in addition, host-directed antivirals that target key steps in the viral lifecycle and are highly conserved and broadly expressed are also likely to emerge across cell types. one example is the endosomal acidification blocker hydroxychloroquine which indeed scored as antiviral in both cell types ( , , ) . in total, we identified three drugs as antiviral in both screens. we performed dose-responses in vero cells against the candidates from the huh . screen. we found that additional compounds were antiviral in vero cells with a si> , azd , bix , ebastine, mg- , and wye- , albeit at higher concentrations ( figure s ). however, the majority of the antivirals that were validated in huh . cells were not active in vero cells. we next focused on lung epithelial models as these are the most relevant to human infections. we found that a number of lung-derived epithelial cell lines were refractory to infection (eg a , calu- , nci-h , cfbe o). however, we found that calu- cells, that have been shown to be permissive for many coronaviruses including sars-cov- , were highly readily infected (fig a) ( , , ) . we optimized assays using calu- cells and tested their sensitivity to remdesivir and hydroxycholorquine. as expected, we found that while the direct acting antiviral remdesivir was antiviral; however, hydroxychloroquine had little or no activity in calu- cells (fig b) . this led us to test the antiviral activity of a panel of chloroquine derivatives and we found that none of these had activity against sars-cov- (fig c) , while these compounds are antiviral in both vero cells and huh . cells (fig d) . this suggests that there are major differences in the requirement for endosomal acidification during infection of sars-cov- in lung epithelial cells. endosomal acidification is thought to be required for sars-cov- entry to maintain the low ph necessary for endosomal cysteine protease activity required for priming spike for membrane fusion ( ) . consistent with the requirement for acidification in vero and huh . , the cathepsin inhibitor z-fa-fmk emerged as antiviral in both cell types (fig d, fig e) . we tested z-fa-fmk in calu- cells and found that it had no antiviral activity (fig d) , consistent with a lack of a requirement for endosomal acidification. recent studies found the plasma membraneassociated serine protease, tmprss , can prime the viral glycoprotein for entry in lung epithelial cells ( ) . therefore, we tested the role of tmprss by treating cells with the inhibitor camostat. we found that camostat was antiviral in calu- cells but had no activity in either vero or huh . cells (figure e -f) ( ) . moreover, the main endosomal kinase phosphatidylinositiol- -phosphate/phosphatidylinositol -kinase, pikfyve, promotes internalization of diverse viruses and was recently shown to impact entry of coronaviruses including sars-cov- in hela cells ( ) . using the pikfyve inhibitor apilimod, we found that pikfyve promotes infection of sars-cov- in huh . and vero cells, with little importance calu- cells ( fig s ) . these data suggest that the entry pathway used by sars-cov- is cell-type specific. to determine which of the antiviral candidates validated in huh . cells also had antiviral activity in calu- cells we performed dose-response studies. we found that drugs were antiviral against sars-cov- in calu- cells with a selectivity index greater than (fig ) . these include: two drugs with unclear targets (salinomycin, y- ), kinase inhibitors (azd , bemcentinib, dacomitinib, wye- ), histamine receptor inhibitor (ebastine), iron chelator dp mt, and the cyclophilin inhibitor cyclosporine. many kinase inhibitors were quite potent, suggesting an important role in intracellular signaling for infection. the other drugs tested in calu- with a si< are shown in fig s . the full table of candidates from the huh . screen with ic , cc and si are shown in figure s . cyclosporine is an fda approved generic drug that is readily available and showed a submicromolar ic with high selectivity in both huh . and calu- cells (fig , fig , fig s ) . cyclosporine binds cyclophilin a and prevents activation of the phosphatase calcineurin which is required for the nuclear translocation of the nuclear factor of activated t cells (nfat) ( ) ( ) ( ) . inhibition of this pathway in t cells is used as an immunosuppressant ( ) . cyclosporins have been shown to have antiviral activity against a wide variety of viruses, including other coronaviruses ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the activity of cyclosporine against previously studied coronaviruses is cyclophilin-dependent and independent of calcineurin ( , ) . we set out to perform initial structure-activity relationships (sar) and to determine if this activity was through its inhibition of cyclophilin or inhibition of calcineurin. for these studies, we obtained a panel of cyclosporine analogs including cyclosporin a, cyclosporin b, cyclosporin c, cyclosporin h and isocyclosoporin a ( ). we found that isocyclosporin a, cyclosporin a, cyclosporin b, and cyclosporin c were active with increasing ic s (fig a-c) . cyclosporine h, shows only weak binding to cyclophilin a, and has no immunosuppressant activity, as it does not inhibit the phosphatase activity of calcineurin ( , ) . cyclosporin h has a log reduction in activity compared to cyclosporine, but retains some activity. psc is a non-immunosuppressant derivative of cyclosporine that does not inhibit calcineurin, has a similar activity to cyclosporin c( ). tmn is a cyclophilin a inhibitor that is times more potent than cyclosporine a in inhibiting the prolyl isomerase activity of cyclophilin a ( ) . we found the latter to lack antiviral activity, suggesting that the enzymatic activity of cyclophilin a is dispensable. we further validated that cyclosporine is antiviral in both cell types by performing rt-qpcr (fig d) . in addition, nim is another non-immunosuppressive cyclosporine derivative of cyclosporine, and we found that it is potently antiviral (fig e) , further suggesting that the antiviral activity is cyclophilin-dependent and separable from calcineurin. strikingly, the activity of this panel of drugs are similar in the two cell lines. these data suggest that cyclosporine has the same target and mechanism-of-action. we also found that none of these drugs are antiviral in vero cells ( figure s ). to further assess the mechanism by which cyclosporine is antiviral, we tested fk , an inhibitor of calcineurin. fk binds the related immunophilin fkbp, rather than cyclophilin a, to block the phosphatase activity of calcineurin, and thus is also a potent immunosuppressant ( ) . we found that fk has no activity against sars-cov- (fig a-c) . moreover, since one of the major targets of calcineurin is the activation of nfat, we also tested whether an nfat inhibitor impacted viral infection ( ) . we found that the nfat inhibitor had no effect on infection (fig a-c) . altogether, we found that cyclosporins are potent antivirals against sars-cov- in lung epithelial cells, and that this activity is independent of calcineurin and nfat. the emergence of sars-cov- has led to devastating global morbidity and mortality, creating an immediate need for new therapeutics and vaccines. repurposing existing drugs can allow for rapid deployment of therapeutics that have already been tested in humans ( ) . remdesivir was developed against the ebola virus rna-dependent rna polymerase, and was also found to have robust activity against sars-cov- ( ) . importantly, we found that remdesivir is active against sars-cov- across cell types. chloroquine and hydroxychlorquine have been used for decades to treat malaria and have been shown to have in vitro antiviral activity against sars-cov- ( , ) . however, we find that this antiviral activity is cell-type specific. lung epithelial cells are resistant to these drugs, and this may explain the lack of efficacy seen in many trials ( ) . to determine if there are additional drugs that are active against sars-cov- in vitro, we screened a repurposing library that includes ~ fda approved drugs and ~ additional drugs that have been tested in humans. repurposing can be used to reveal new and similar pathways and targets, but also the time and monetary investment associated with repurposing is potentially less since these drugs often bypass phase- trials ( , ) . initial screens in vero cells yielded few active drugs, leading us to pursue a screen in human huh . cells, a transformed hepatocyte line deficient in innate immune signaling. using this model system, we identified drugs, and validated with dose-response assays. this includes many drugs that were previously shown to have activity against other coronaviruses (tetrandrine, cepharanthine, cyclosporine, alixostatin, mg , salinomycin), and sars-cov- (salinomycin, tetrandrine, cepharanthine, cyclosporine, ebastine) ( , , , , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the drugs fall into distinct classes and most have known targets. however, two drugs that were active across cell types, salinomycin and y- , do not have clear targets. salinomycin, is a polyether antibiotic and chemotherapy drug, that has been shown to be antiviral against many viruses, including coronaviruses ( , ( ) ( ) ( ) . salinomycin was also identified in a vero cell screen ( ) . mechanistically, some studies have suggested that salinomycin is an ionophore that can attenuate viral entry by disrupting the acidification of the endosome ( ) . other studies have implicated salinomycin in er stress ( ) . studies in mice have shown antiviral activity against influenza ( ) . salinomycin has also been characterized as an activator of autophagy, which may influence sars-cov- infection ( , ) ( ). y- is a phenylpyrazoleanilide immunomodulatory agent that has been shown to inhibit il- production by t cells and has activity in monkeys ( ) . interestingly, treatment with y- is associated with decreased il- production, a cytokine that is thought to be highly expressed in sars-cov- infection ( ) ( ) ( ) . however, it is unclear how y- could attenuate sars-cov- in non-immune cells. ebastine is a potent h -histamine receptor antagonist, used for allergic disorders outside of the us, particularly in asia ( ). we found that ebastine is antiviral in all three cell types, although -fold less active in vero cells ( ) . ebastine is orally available with few side effects and there are there clinical trials underway in china testing whether ebastine can impact covid- outcomes ( ) . since other h -histamine receptor antagonists were not active, it is unclear why this particular agent is more effective at inhibiting sars-cov- infection. interestingly, ebastine and its active metabolite, carebastine, are reported to inhibit expression of il- , while many other h -histamine receptor antagonists do not ( , ) . we also identified protease inhibitors as antiviral in huh . cells. two cysteine protease inhibitors, z-fa-fmk and mg- , had activity in both vero and huh . cells. none of the protease inhibitors were active in calu- cells. this observation suggests that they are not targeting the viral proteases. consistent with this, z-fa-fmk is an inhibitor of cathepsins which are required for sars-cov- entry in cells where endosomal proteases are required for spike cleavage, and thus we observe no requirement in calu- cells where tmprss is required for infection ( , , ( ) ( ) ( ) .this has important implications in diverse sars-cov- studies where there may be cell-type specific requirements for different steps in the replication cycle. we also identified two inhibitors against the cellular histone methyltransferase g a as antiviral in huh . cells. however, these drugs were not active in calu- cells, suggesting that there are cell type specific requirements. am is a selective cannabinoid cb receptor agonist that we found was antiviral in huh . cells. gw x, another cb agonist that has a -fold higher ec , was not active. moreover, dose-response studied found that am is not active in either vero or calu- cells. cepharanthine and tetrandrine are both bis-benzylisoquinoline alkaloids produced as natural products from herbal plants ( ) . tetrandrine, a traditional chinese medicine and calcium channel blocker, has been shown to antagonize calmodulin. it has anti-tumor and antiinflammatory effects, and can effectively inhibit fibroblasts, thereby inhibiting pulmonary fibrosis ( , ) . multiple studies have suggested that tetrandrine has antiviral activity, including against dengue virus and herpes simplex virus ( , ) . tetrandrine has also been shown to inhibit entry of ebola virus into host cells in vitro and showed therapeutic efficacy against ebola in preliminary studies on mice ( ) . currently, there is an ongoing clinical trial using tetrandrine in covid- patients to improve pulmonary function ( ) . cepharanthine is reported to have antiinflammatory and immunoregulatory properties and is used to treat a variety of acute and chronic conditions outside of the us ( ) . both cepharanthine and tetrandrine were previously shown to have antiviral activity against the human coronavirus oc and in recent studies on sars-cov- in vero cell screens ( , , ) . while both of these molecules were antiviral in our huh . screen, neither were active in calu- cells. this may suggest that they are modulating endosomal entry pathways. we identified few metabolic regulators. dp mt is a potent iron chelator that we found to be antiviral against sars-cov- in huh . and calu- cells ( ) . a clinical trial with the iron chelator deferoxamine is underway (nct ). however, other iron chelators in our library, deferasirox and deferiprone, were not identified as antiviral making the mechanism of action unclear. we identified several kinase inhibitors as antivirals against sars-cov- . frax is a p activated kinase (pak) inhibitor that is antiviral in huh . cells, but only modestly impacted infection of calu- cells ( ) . other pak inhibitors were not identified in our screens. pak is required for entry by many viruses( ). pd is a potent wee and chk inhibitor that is antiviral in huh . cells, but shows strong toxicity in calu- cells ( ). we also found three mtor inhibitors, azd , pf- , and wye- are antiviral against sars-cov- in huh- and calu- cells. these are highly potent atp competitive mtor inhibitors that target both torc and torc . in our library, none of the rapamycin analogs that selectively inhibit mtorc were active. we also identified two potent selective and irreversible inhibitors of egfr, dacomitinib and naquotinib. the other egfr inhibitors showed no activity in huh . cells. importantly, dacomitinib is a potent antiviral in calu- cells. it is unclear if the target is indeed egfr, but for many viruses egfr activation promotes viral entry which may also be the case for sars-cov- ( - ). cyclosporine is a commonly used immunosuppressant that binds cyclophilin a and inhibits the calcium-dependent phosphatase calcineurin which is required for the nuclear translocation of the nuclear factor of activated t cells (nfat) ( ) ( ) ( ) ( ) ) . inhibition of this pathway in t cells is used as an immunosuppressant. we found that cyclosporine is active in both huh . and calu- cells, but has no activity in vero cells nor did the cyclosporine analogs. a recent screen in vero cells did find activity with cyclosporine against sars-cov- ( ) . cyclophilin a is a ubiquitously expressed peptidyl-prolyl cis-trans isomerase ( ). cyclophilin a and other cyclophilins have chaperone-like activity and take part in protein-folding processes ( ) . cyclophilin a has been shown to be an important cellular factor that facilitated many diverse viral infections. this includes human immunodeficiency virus type (hiv- ), influenza virus, hepatitis c virus (hcv), hepatitis b virus (hbv), vesicular stomatitis virus (vsv), vaccinia virus (vv), severe acute respiratory syndrome coronavirus (sars-cov) and rotavirus (rv) ( - , , ) . the coronaviruses hcov- e, hcov-nl , fpiv, mouse hepatitis virus (mhv), avian infectious bronchitis virus, and sars-cov have been found to be attenuated by cyclosporin a ( , , ) . cyclosporine and its non-immunosuppressive derivatives can inhibit replication of a number of viruses including some coronaviruses. in most cases the responsible cyclophilin is cypa ( , ), but cypa and cypb were found to be required for fcov replication ( ) . for hcov-nl , and hcov- e, cyclophilin a is required for infection in caco- cells ( ) and huh- . cells respectively ( , ) . it is generally thought that the activity of cyclosporine against coronaviruses is cyclophilin-dependent and independent of calcineurin. we found that a number of cyclosporins were antiviral with similar potencies including cyclosporine, cyclosporin a, cyclosporin b and the metabolic breakdown product of cyclosporin a, isoscyclosporin a. we also found that cyclophilin a is likely required, as cyclosporin h, which is a weak binder had reduced activity. however, the enzymatic activity of cyclophilin a is likely dispensable as tmn was inactive. to further address the role of calcineurin, we tested a non-immunosuppressant derivative of cyclosporine that does not inhibit calcineurin, has a similar activity to cyclosporin c. we also found that fk , a calcineurin inhibitor independent of cyclophilin a, and nfat inhibitors also have no antiviral activity. altogether, we found that cyclosporins are potent antivirals against sars-cov- in lung epithelial cells, and that this activity is independent of calcineurins. nim is a cyclophilin inhibitor independent of calcineurin, and we found that this is highly active in huh . cells, further suggesting that cyclophilin is required for sars-covo- infection. strikingly, the activities of all of these drugs is similar in the two cell lines suggesting the same target and mechanism-of-action and that cyclosporine would block sars-cov- in diverse infected tissues in vivo. one approach would be to use cyclophilin inhibitors that do not have immunosuppressive activity such as nim or others that have been tested for hcv infection (alisporivir (debio- ) and scy- ) ( ) or for hiv infection (nim ) ( ) ( ) . another possibility is to use cyclophilin inhibitors that also target calcineurin (eg. cyclosporine). one of the major complications of covid- is the hyper-inflammatory response and cytokine storm associated with increased immune activation. to prevent hyper-activation, there has been interest in treating covid- patients with immunosuppressants ( ). there are ongoing trials for a variety of agents including anti-il and jak inhibitors, two clinical trials using sirolimus, the fda approved mtor inhibitor, which selectively inhibits mtorc . we find no antiviral activity of sirolimus or other rapamycin derivatives. in contrast, cyclosporin a is an approved immunosuppressant that we found is also antiviral at concentrations achieved in vivo ( ) . therefore, it may be useful to implement clinical trials using cyclosporin a as an immunosuppressant as it would potentially ameliorate symptoms by two mechanisms ( ) . there have been a large number of screens posted in the literature that suggest antiviral activity of several existing drugs (e.g. azithromycin, faviprivir, lopinavir, ribavirin, and ritonavir, tetracycline, etc). these drugs and most screens have been performed in vero cells, with toxicity as read-outs. medicines for malaria venture (mmv) has compiled a list of drugs that has support for antiviral activity against sars-cov- (https://www.mmv.org/mmv-open/covid-box). we tested > of the compounds and find that in addition to the quinolines and drugs found in our screen there are few additional compounds that show activity at less than um. while it is possible that some of these drugs are false negatives in our screens, it is likely that many of these candidates do not have antiviral activity when either measuring viral antigen production or when looking in different cell types. it is very important that identified antivirals be tested for their impact on viral replication more directly. moreover, given the striking differences in sensitivities across cell types it is important to validate the activity of any new antivirals in lung epithelial cells. altogether, these studies highlight the roles of cellular genes in viral infection, cell type differences, and our discovery of nine broadly active antivirals suggest new avenues for therapeutic interventions. we found that of the drugs are antiviral in lung epithelial cells, have been used in humans, of these are fda approved in the us (cyclosporine, dacomitinib, and salinomycin), and ebastine is approved outside of the us. while clinical trials are underway with some of these candidates, additional trials will be needed to determine the efficacy of these antivirals in covid- patients, to inform future treatment strategies. vero e cells and vero ccl were obtained from atcc and were cultured in dmem, supplemented with % (v/v) fetal bovine serum, % (v/v) penicillin/streptomycin, % (v/v) l-glutamax and were maintained at °c and % co . huh . cells were obtained from c. rice (rockefeller) and cultured in dmem, supplemented with % (v/v) fetal bovine serum, % (v/v) penicillin/streptomycin, % (v/v) l-glutamine, and were maintained at °c and % co . calu- cells (htb- ) were obtained from atcc and cultured in mem, supplemented with % (v/v) fetal bovine serum, % (v/v) penicillin/streptomycin, % (v/v) l-glutamine, and were maintained at °c and % co . sars-cov- was obtained from bei (wa- strain). stocks were prepared by infection of vero e cells in % serum plus mm hepes for five days, freezethawed, and clarified by centrifugation (po). titer of stock was determined by plaque assay using vero e cells and were x pfu/ml and . x tcid /ml ( ) . this seed stock was amplified in vero ccl (p ) at . x tcid /ml. all work with infectious virus was performed in a biosafety level laboratory and approved by the institutional biosafety committee and environmental health and safety. infections: cells were plated in well plates ( µl/well) , cells per well for vero, , cells per well huh . , , cells per well calu- . the next day, nl of drugs were added. the positive control remdesivir and the negative control dmso were spotted on each plate. one hour later cells were infected with sars-cov- (vero, moi= ; huh . moi= ; calu- moi= . ) cells were fixed ( hpi vero and huh , , hpi calu- ) in % formaldehyde/pbs for min at room temperature and then washed three times with pbst. cells were blocked ( % bsa/pbst) for minutes and incubated in primary antibody (anti-dsrna j ) overnight at c. cells were washed x in pbst and incubated in secondary (anti-mouse alexa and hoescht ) for h at room temperature. cells were washed x in pbst and imaged using imagxpress micro using a x objective. four sites per well were captured. the total number of cells and the number of infected cells were measured using metaxpress . . cell scoring module, and the percentage of infected cells was calculated. the aggregated infection of the dmso and remdesivir control wells (n= ) on each assay plate were used to calculate z'-factors, as a measure of assay performance and data quality. sample well infection was normalized to aggregated dmso plate control wells and expressed as percentage of control [poc = (%infection sample / average %infection dmso )* ] and z-score [z= (%infection sample -average %infection dmso ) / standard deviation %infection dmso ]in spotfire (perkinelmer). candidate hits were selected as compounds with poc< % and viability > %, compared to vehicle control. candidate drugs were repurchased as powders from selleckchem, medchemexpress, and medkoo and suspended in dmso. drugs were arrayed in -pt dose-response in well plates. infections were performed using screening conditions. dmso (n= ) and µm remdesivir (n= ) were included on each validation plate as controls for normalization. infection at each drug concentration was normalized to aggregated dmso plate control wells and expressed as percentage-of-control (poc=% infection sample /avg % infection dmso cont ). ). a nonlinear regression curve fit analysis (graphpad prism ) was performed on poc infection and cell viability using log transformed concentration values to calculate ic values for infection and cc values for cell viability for each drug/cell line combination. selectivity index (si) was calculated as a ratio of drug's cc and ic values (si = cc /ic ). rt-qpcr: huh . ( , cells/well) or calu- cells ( , cells/well) were plated in well plates. the next day, drugs were added and one hour later infected with sars-cov- (moi= . ). total rna was purified using trizol (invitrogen) followed by rna clean and concentrate kit (zymo researc) hpi for huh . or hpi for calu- . for cdna synthesis, reverse transcription was performed with random hexamers and moloney murine leukemia virus (m-mlv) reverse transcriptase (invitrogen). synthesized rna was used as a standard (bei). gene specific primers to sars-cov- (wuhan v , nsp ) and sybr green master mix (applied biosystems) were used to amplify viral rna and s rrna primers were used to amplify cellular rna using the quantstudio flex rt-pcr system (applied biosystems). relative quantities of viral and cellular rna were calculated using the standard curve method ( ) . viral rna was normalized to s rna for each sample (wuhan v / s). wuhan-v _forward s rrna_forward '-aacccgttgaaccccatt- ' s rrna reverse '-ccatccaatcggtagtagcg- ' we thank s. weiss and y. li for sharing sars-related coronavirus , isolate usa-wa / (obtained from the centers for disease control and bei resources). we thank bei resources for quantitative sars-cov- rna. we thank m. diamond and s. hensley for providing anti-spike antibody (cr ), c. coyne for j antibody, m. diamond for oligo sequences. we thank e. grice for hacat cells. we thank c. kovacsics for biosafety support. we thank the cherry lab, the high-throughput screening core, david roth, and john epstein for discussions. we thank timothy wells and medicines for malaria venture for helpful discussions and compounds. we thank the nih, dean's innovation fund, linda and laddy montague, bwf for funding. origin and evolution of pathogenic coronaviruses coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus sars and mers: recent insights into emerging coronaviruses a new coronavirus associated with human respiratory disease in 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resistance in vitro cyclophilin a and viral infections feline coronavirus replication is affected by both cyclophilin a and cyclophilin b cyclophilin inhibition as potential therapy for liver diseases sars-cov- pandemic : time to revive the cyclophilin inhibitor alisporivir cytokine release syndrome in severe covid- cyclosporin. a review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in immunoregulatory disorders covid- and calcineurin inhibitors: should they get left out in the storm? a standard curve based method for relative real time pcr data processing key: cord- - mx o eb authors: wang, yilong; liu, rongxian; lu, mijia; yang, yingzhi; zhou, duo; hao, xiaoqiang; zhou, dongming; wang, bin; li, jianrong; huang, yao-wei; zhao, zhengyan title: enhancement of safety and immunogenicity of the chinese hu measles virus vaccine by alteration of the s-adenosylmethionine (sam) binding site in the large polymerase protein date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: mx o eb the live-attenuated measles virus (mv) vaccine based on the hu strain has played a significant role in controlling measles in china. however, it has considerable adverse effects that may cause public health burden. we hypothesize that the safety and efficacy of mv vaccine can be improved by altering the s-adeno- sylmethionine (sam) binding site in the conserved region vi of the large polymerase protein. to test this hypothesis, we established an efficient reverse genetics system for the rmv-hu strain and generated two recombinant mv-hu carrying mutations in the sam binding site. these two mutants grew to high titer in vero cells, were genetically stable, and were significantly more attenuated in vitro and in vivo compared to the parental rmv-hu vaccine strain. importantly, both mv-hu mutants triggered a higher neutralizing antibody than rmv-hu vaccine and provided complete protection against mv challenge. these results demonstrate its potential for an improved mv vaccine candidate. measles virus (mv) is an enveloped virus with a non-segmented, negative-sense (nns) rna genome in the family paramyxoviridae, order mononegavirales . in developing countries, measles is still a leading cause of mortality in children (griffin and oldstone, ; tangy and naim, ) , though vaccination is an effective, economical, and safe way to prevent outbreaks (bester, ; de vries et al., ) . in early , a live-attenuated vaccine based on the hu strain of mv was developed and is currently widely used for immunization in all provinces of china (zhang et al., ) . while this vaccine is efficacious, it has associated adverse effects. many vaccinated infants and children in china experienced side effects ranging from skin rashes, itching, swelling, and to high fever (bester, ; shu et al., ) . additionally, outbreaks of measles have been increasing significantly in the past a few years in china, particularly the increasing proportion of adult and infant cases (ma et al., ; zhang et al., ) . the infected adults had received measles vaccination during childhood; still remain susceptible to infection with the measles virus, as the population immunity against measles after vaccination gradually reduces with time (abad and safdar, ; gao et al., ; ma et al., ; zhang et al., ) . thus, there is an increasing urgency to develop a safer, more efficient mv vaccine for eradication of measles in china. reverse genetics system has been established for many nns rna viruses including the vesiculovirus, morbillivirus, respirovirus, and pneumovirus (neumann et al., ) . similar to other nns rna viruses, the minimal machinery for mv transcription and replication is the ribonucleoprotein (rnp) complex, which consists of the nucleocapsid (n)-rna template tightly associated with the rna-dependent rna polymerase, the large (l) protein and the phosphoprotein (p). assembly of replication-competent rnps is essential to the rescue of nns rna viruses (bukreyev et al., ; clarke et al., ; garcin et al., ; gassen et al., ; jin et al., ; lawson et al., ) . this can be achieved by co-transfection of a plasmid encoding a fulllength antigenomic cdna together with plasmids encoding n, p, and l genes. previously, several groups have already successfully rescued infectious mv from cdna clones (duprex et al., ; kovacs et al., ; nakatsu et al., ; parks et al., ; radecke et al., ; sidhu et al., ) . the reverse genetics system can facilitate the rational design of safer, more efficient measles vaccine candidates. the l protein of nns rna viruses possesses the majority of enzymatic activities for transcription and replication (ferron et al., ; poch et al., ; whelan et al., ) . during transcription, nns rna viruses synthesize mrnas that are capped and methylated at the 'end and polyadenylated at the ' end. recent studies have shown that the entire mrna capping and methylation machinery of nns rna viruses is distinct from their host (ferron et al., ; furuichi and shatkin, ; ogino and banerjee, ; zhang et al., ) . using vesicular stomatitis virus (vsv) as a model, it was found that vsv mrna capping is catalyzed by an rna:gdp polyribonucleotidyltransferase (prntase) in the l protein that transfers a monophosphate rna onto a gdp acceptor (li et al., ; ogino and banerjee, ) . the mrna cap methylation in nns rna viruses is also unusual in that a single region in the l protein catalyzes both guanine-n- (g-n- ) and ribose ′-o ( ′-o) methylation (li et al., ; rahmeh et al., ) . thus, mrna cap formation is an excellent target for development of antiviral drugs and live vaccine candidates for nns rna viruses. based on the sequence alignments, the l protein contains six conserved regions (cr) numbered i to vi. recent studies showed that cr v of the l protein possesses an mrna capping enzyme whereas cr vi is responsible for mrna cap methyltransferase (mtase) activity (li et al., ; ogino et al., ) . it was shown that mutations to the capping enzyme were lethal to the virus. however, mutations to mtase region yielded recombinant viruses that were attenuated in vitro and in vivo. this suggests mrna cap mtase is a novel target for rational design of live attenuated vaccines for nns rna viruses. this novel concept has recently been tested in several nns rna viruses including vsv, avian metapneumovirus (ampv), human metapneumovirus (hmpv), and rabies virus (rabv) (li et al., ; sun et al., ; tian et al., ; zhang et al., ) . it was shown that recombinant viruses lacking mtase activity are highly attenuated in vitro and in vivo, yet retain optimal immunogenicity. we hypothesized that engineering mutations to the mtase region of mv l protein would lead to further attenuation of the current live attenuated vaccine strain, enhancing the safety of mv vaccine. to test this hypothesis, we established a robust reverse genetics system based on a chinese mv vaccine strain mv-hu- , allowing us to recover recombinant mv in bhk cells stably expressing t rna polymerase (xu et al., ; zhang et al., ) . subsequently, two recombinant mvs with amino acid (aa) substitutions in the s-adenosylmethionine (sam) binding site of l protein (rmv-hu -g a and rmv-hu -g a) were successfully recovered. these two mtase-defective mutants had delayed replication kinetics, grew to high titers, and were genetically stable through passages in cell culture. both mv mutants were significantly more attenuated in vitro and in vivo compared to the parental vaccine strain. interestingly, both mutants induced significantly higher neutralizing antibody titers compared to the parental fig. . construction of a full-length cdna clone for mv-hu . the t promoter, ′ and ′ non-coding termini (nct), antigenomic hdv ribozyme and t terminator were assembled in several rounds of fusion pcr, and inserted into pyes- using a "seamless" cloning strategy, resulted in the construction of p -mv(+) (a). eight overlapping fragments containing the full-length mv genome were assembled into p -mv(+), creating pyes-mv(+) (b). a spontaneous mutation (c to u) in the h gene that distinguishes the lab-propagated parental virus and rescued recombinant virus was marked by "*" (b). virus. these results demonstrate that alteration of sam binding sites in mv l protein enhances both the safety profile and the immunogenicity of the mv vaccine. thus, mrna cap mtase can serve a novel approach for rational design of a safer and more efficacious mv vaccine. a full-length cdna clone of mv strain hu , pyes-mv(+), was constructed by a novel methodology using the geneart™ high-order genetic assembly system. the full-length cdna clone of mv-hu was successfully assembled by a single step ligation, without the need for restriction endonucleases. the , -nt antigenomic mv cdna was cloned under the control of a t rna polymerase promoter, a hepatitis delta virus (hdv) ribozyme sequence, and a t terminator ( fig. ; table ). to recover infectious mv, bhk-sr -t cells stably expressing t rna polymerase were co-transfected with full-length cdna clone pyes-mv(+) and the support plasmids expressing ribonucleoprotein (pt -hu -n, pt -hu -p, and pt -hu -l). three days post-transfection, cell monolayers were trypsinized and co-cultured with fresh vero cells. mv-induced syncytia were observed - days later ( fig. a , b and c). the successful recovery of rmv-hu was further confirmed by detection of n protein expression in vero cells infected with the rescued rmv-hu by an immunofluorescence assay (fig. d ). when extensive syncytia were observed, cells were harvested and the supernatants were used for further passage in vero cells. after - passages, recombinant mv was plaque purified, and a large stock of virus was prepared. there is a spontaneous mutation (c to u) at nucleotide (nt) position within the h gene of the lab-propagated parental virus, which is different from the published hu sequence (genbank accession no. fj ). this site in pyes-mv(+) was mutated back to c by site-directed mutagenesis with pcr, which distinguished the parental virus in the lab but is identical with the published sequence. to confirm the recovered recombinant virus (rmv-hu ) originated from pyes-mv(+) and not from cross-contamination of the parental mv-hu grown in our laboratory, a region of the rmv h gene was amplified by rt-pcr and sequenced. the result showed that rmv-hu contained the "c" mutation. having the establishment of robust reverse genetics for rmv-hu , we next tested the hypothesis that the rmv-hu vaccine strain can be further attenuated by alteration of the sam binding site in mv l protein. previously, this strategy was used in the rational design of live attenuated vaccine candidates for vsv, ampv, and hmpv (li et al., ; sun et al., ; zhang et al., ) . the sam-dependent mtase superfamily typically contains a conserved g-rich motif for binding the sam molecule, the methyl donor for rna methylation (mcilhatton et al., ; schluckebier et al., ) . sequence alignment revealed that a gxgxgx motif was conserved in cr vi of the l proteins of all paramyxoviruses and most of the mononegavirales (fig. ) (li et al., ; mcilhatton et al., ; poch et al., ; zhang et al., ) . sequence analysis found that aa residues corresponding to the gxgxgx motif of the mv l protein includes g , g , and g . therefore, these amino acids were individually mutated to alanine in an infectious cdna clone of mv, pyes-mv(+ ). three recombinant mv clones with a single point mutation (g a, g a or g a) in their sam binding sites were constructed. using the reverse genetics system, two recombinant mvs, rmv-hu -g a and rmv-hu -g a, were successfully rescued and viral titer gradually increased when they were passaged in vero cells. the rmv mutants were confirmed by detection of n protein expression in vero cells infected with the rescued rmv mutants by immunofluorescence (figs. e and f). the rmv-hu -g a mutant was viable, but it grew poorly in vero cell and further passages of this mutant did not increase viral titer (data not shown). next, rmv-hu -g a and rmv-hu -g a were plaque purified. the sizes of virus-induced plaques differed between the rescued parental and mutant viruses. as demonstrated in fig. , after days of incubation, the parental rmv-hu formed plaques were . ± . mm in diameter, whereas the average plaque for rmv-hu -g a and rmv-hu -g a was significantly smaller ( . ± . mm and . ± . mm, respectively; p < . ). this suggests that the two mv mutants likely had impaired growth kinetics that caused the plaque sizes to be reduced. finally, the entire genome of each mv mutant was amplified by rt-pcr and sequenced. result showed that each mutant retained the desired mutation. in addition, no other mutations were found in the genome. we next compared the replication kinetics of the rmv-hu mutants and the parental virus in vero cells in the time course of h after infection (fig. ). parental rmv-hu reached a peak titer ( . ± . log pfu/ml) at h post-inoculation (hpi), while peak titers for the two mutants at hpi. importantly, rmv-hu -g a was delayed in replication but reached a peak titer of . ± . log pfu/ml at hpi, which was comparable to the parental virus (p > . ). the peak titer achieved by rmv-hu -g a was . ± . log pfu/ml at hpi, which was significantly lower than that of parental rmv-hu at hpi (p < . ). both mv mutants had delayed cytopathic effects (cpe) compared to the parental virus. the parental rmv-hu developed extensive cell-to-cell fusion and large syncytia at hpi and reached maximum cpe at hpi, whereas mutants rmv-hu -g a and rmv-hu -g a had a delay in formation of syncytia and reached maximum cpe at hpi (fig. ) . these data suggest that rmvs carrying mutations in the sam binding site were more attenuated in vero cells than the parental mv vaccine strain. to investigate whether rmv-hu -g a and rmv-hu -g a were genetically stable in vitro, each virus was passaged in vero cells for times. the mutated region in the l gene was sequenced for each of the first passages. virus in each passage retained the desired mutation. at passage , the entire genome of each mutant was sequenced, showing no additional mutations in the genome. table sequences of the oligonucleotides used for pcr. sequence ( ′− ′) virology ( ) - . . rmv-hu carrying mutations in the sam binding site are more attenuated in vivo than the parental vaccine strain four-to-six-week-old specific-pathogen-free (spf) cotton rats were inoculated intranasally with parental and mutant rmv-hu in order to determine their replication in vivo. no clinical symptoms of respiratory tract infection were found in cotton rats inoculated with any of the rmvs. at day post-inoculation, cotton rats were terminated, and viral titer in the lungs was determined ( table ). the parental virus replicated efficiently in lungs with an average titer of . log pfu/g lung tissue. recombinant rmv-hu -g a had an average titer of . log pfu/g lung tissue, which was significant lower than rmv-hu (p < . ). however, rmv-hu -g a was the most attenuated mutant. only out of cotton rats had detectable viral titer in the lung with a titer of . log pfu/g. these results show that the two mutant rmvs were more attenuated in viral replication in vivo compared to the parental vaccine strain. to ensure that each mutant was stable in vivo, total rna was extracted from each lung sample, and the regions harboring mutations were amplified by rt-pcr. the samples from each animal were sequenced, respectively. the result showed that the desired mutation was retained in rmv-hu -g a or rmv-hu -g a from each animal. no additional mutations were detected in the sequenced region. . . rmv-hu mutants induce higher neutralizing antibodies than the parental vaccine strain and provide complete protection against mv challenge the immunogenicity of rmv-hu mutants was assessed in cotton rats. briefly, - -week-old spf cotton rats were intranasally inoculated with . × pfu of each mv, and were challenged with . × pfu of rmv-hu at week post-immunization. the two mutant rmvs induced high levels of neutralizing antibodies as early as week after vaccination, and antibodies gradually increased from weeks - . however, antibodies produced by the parental rmv-hu peaked at week , and declined during weeks and . overall, the antibodies induced by rmv-hu -g a and rmv-hu -g a were comparable to those generated by wild-type rmv-hu at weeks - (p > . ) (fig. a) . however, at week , neutralizing antibodies induced by rmv-hu mutants were significantly higher than those from parental rmv-hu (p < . ; fig. b ). this suggests that rmv-hu mutants were more immunogenic compared to the parental vaccine strain. at week post-vaccination, cotton rats were challenged with . × pfu of rmv-hu and all cotton rats were terminated at day post-challenge. no infectious virus was detected in the lung tissue of any of the vaccinated cotton rats (table ). in contrast, an average titer of . ± . log pfu/g was detected in lung tissue from unvaccinated but challenged controls. these results show that rmv-hu -g a and rmv-hu -g a provide complete protection from mv challenge. in this study, we successfully generated two recombinant measles viruses with amino acid substitutions in the sam binding site of l protein and examined the effects of these mutations on viral replication, safety, and immunogenicity. we found that both rmv-hu -g a and rmv-hu -g a were significantly more attenuated compared to parental rmv-hu , the widely used vaccine in china. rmvs carrying mutations in the sam binding site were genetically stable, formed significantly smaller viral plaques, and had delays in cpe and replication kinetics. recombinant rmv-hu -g a grew to high titer in vero cells that was comparable to rmv-hu but exhibited significantly more attenuation in cotton rats. recombinant rmv-hu -g a grew to a relatively lower titer in vero cells but had a greater degree of attenuation in cotton rats compared to rmv-hu -g a. both recombinant viruses triggered significantly higher neutralizing antibody compared to rmv-hu , and provided complete protection against mv challenge. this indicates that alteration of sam binding site in mv l protein enhances the safety and immunogenicity of the rmv-hu vaccine strain. we established a more efficient method to assemble a full-length cdna clone of mv-hu without using restriction endonucleases. the mv genome was divided into eight overlapping fragments and assembled into a full-length plasmid using the geneart™ high-order genetic assembly system. the traditional method for assembly of an infectious cdna requires multiple cloning steps involved in restriction enzyme digestion and ligation, which are time consuming, labor extensive, and technically challenging. the traditional cloning strategy also often leads to some unexpected deletions, insertions, and mutations in the viral genome, which hamper the subsequent virus rescue. our assembly strategy was highly efficient, allowing us to obtain full-length cdna clones in a single step. previously, vaccinia virus vtf- or mva-t providing t rna polymerase had often been used to rescue mv in reverse genetics system. however, there was difficulty in separating the rescued mv and the helper viruses. bhk cells stably expressing t rna polymerase, instead, were used to rescue hmpv and bovine respiratory syncytial virus (buchholz et al., ; zhang et al., ) . in our study, bhk-sr -t cells were co-transfected with a plasmid expressing antigenomic mv cdna and support plasmids expressing the mv n, p, and l proteins, allowing for efficient recovery of infectious mv in a vaccinia virus-free cell system. the primary advantage of this system is the elimination of the potential contamination by the vaccinia virus. this rescue system was highly efficient as we were able to recover many mutants in the cr vi of l protein including the two recombinant viruses with aa substitutions in the sam binding site reported in this study. a live attenuated vaccine is a very promising vaccine for most human paramyxoviruses, as it does not cause enhanced lung diseases upon re-infection by the same virus. a live-attenuated vaccine based on the hu strain of mv has been developed and is widely used for immunization in chinese infants and children (zhang et al., ). however, epidemiological study showed that this vaccine still causes some adverse effects that may cause public health burden. in addition to the safety issue, measles outbreaks have been increasing in recent years, likely due to gradual reducing of the population immunity against measles after vaccination with time (abad and safdar, ; gao et al., ; ma et al., ; zhang et al., ) . in this study, we sought to improve the safety and efficacy of current mv vaccine by mutating the sam binding site in the l protein. using a robust reverse genetics for rmv-hu , rmv-hu carrying mutations in the sam binding site, rmv-hu -g a and rmv-hu -g a, were successfully rescued. these rmv-hu mutants produced smaller plaques, had delayed growth kinetics, and had delayed syncytia formation compared to parental rmv-hu . clearly, both mutants were significantly more attenuated in vero cells than the parental rmv-hu . in cotton rats, rmv-hu -g a had a significantly lower viral titer in the lungs than rmv-hu (p < . ). recombinant rmv-hu -g a was even more attenuated, as only out of inoculated cotton rats had detectable viral titer in the lung. despite the high attenuation phenotype, rmv-hu -g a grew to high titer compared to parental vaccine virus in vero cells, the who approved cell line for vaccine production. although rmv-hu -g a grew to a slightly lower titer ( . log less) in vero cells, it is still economically feasible for vaccine production. another advantage of using rmv-hu -g a is that it had a greater degree of attenuation in vitro and in vivo compared to rmv-hu -g a. interestingly, both mutants triggered a higher level of neutralizing antibodies than parental rmv-hu , suggesting their greater immunogenicity. finally, cotton rats vaccinated with both mutants were completely protected from the mv challenge. thus, recombinant rmv-hu carrying mutations in the sam binding site are potentially improved vaccine candidates for mv. a novel finding is that recombinant rmv-hu carrying mutations in the sam binding site triggered a higher neutralizing antibody compared to the parental rmv-hu strain. although the detailed mechanism is not explored in this study, it is possible that these mv mutants may trigger a higher innate immunity, which in turn triggered a more robust adaptive immunity. in fact, it was shown that coronavirus lacking '-o methylation significantly enhanced type i interferon response, which is another advantage of using viral mrna cap mtase as a target in developing live vaccine candidates. previously, it was found that two pneumoviruses (ampv and hmpv) carrying mutations in the sam binding site in cr vi of l protein were specifically defective in ribose ′-o methylation but not g-n- methylation, and were significantly attenuated but retained wild-type levels of immunogenicity. in addition, it was found that all ′-o mtase-defective hmpvs were highly sensitive to ifn-α and ifn-β treatment (sun et al., ; zhang et al., ) . given the fact that the mtase domain is highly conserved in l proteins of all nns rna viruses, the general mechanism of attenuation of viruses that lack ′-o methylation may be similarly conserved in all nns rna viruses. future experiments should investigate the mechanisms by which mv mutants enhance immunogenicity. we have established a novel and efficient strategy for assembly of a full-length cdna clone of mv-hu and established an efficient vaccinia virus-free reverse genetics system for mv-hu . we generated two recombinant mv-hu carrying mutations in the sam binding site, which not only grew to high titer in vero cells and were genetically stable but also were significantly more attenuated and immunogenic compared to the currently used chinese mv vaccine strain. these two recombinant viruses may serve as improved vaccine candidates for mv. vero cells (african green monkey, atcc-ccl- ) and bhk-sr -t cells (kindly offered by apath, llc, brooklyn, ny) were grown in dulbecco's modified eagle's medium (dmem; life technologies) supplemented with % fetal bovine serum (fbs). the chinese hu vaccine strain of mv (obtained from dr. yiyu lu, zhejiang cdc) was passaged in vero cells. viral rna was extracted from µl of mv-hu using an rneasy mini-kit (qiagen), and reverse-transcribed using super script® iii reverse transcriptase (invitrogen) and random primer mix (neb). the genome was amplified in eight overlapping fragments by q ® high-fidelity × master mix (neb), using eight pairs of mv-specific primers (table ) , and cloned into the peasy-blunt vector (transgen) according to the manufacturer's instructions. the resultant eight plasmids containing the full-length mv-hu genome (peasy-n, peasy-p, peasy-m , peasy-m , peasy-f, peasy-h, peasy-l , peasy-l ) were sequenced, and found to be identical with the published sequence of mv-hu (genbank accession no. fj ), except for a single point change (c to u) at nt within the h gene. this mutation in peasy-h was corrected by site-directed mutagenesis with specific primers ( table ). the resultant plasmid was named peasy-h-m. several rounds of amplification and "in-fusion" pcr were used to assemble five fragments [the t promoter, mv ′ and ′ non-coding termini (nct) ( ′− nt and ′− nt, respectively) were amplified from peasy-n and peasy-l , respectively, hepatitis delta virus (hdv) ribozyme ( nt of anti-genomic hdv sequence), and the t terminator], and subsequently inserted them into the pyes- plasmid using the geneart™ seamless cloning and assembly kit (invitrogen; fig. a ), creating plasmid p -mv(+). the primer sequences and approaches used in the pcr assays are available upon request. the ten fragments were successfully assembled into a full-length cdna clone using the geneart™ high-order genetic assembly system according to the manufacturer's manual (fig. b) , creating plasmid pyes-mv(+). eight mv-hu genomic fragments were amplified with specific primers from peasy-n, peasy-p, peasy-m , peasy-m , peasy-f, peasy-h-m, peasy-l , and peasy-l . the p -mv(+) insert (containing the t promoter, mv ′ and ′ ncts, hdv ribozyme, and t terminator) was divided into two fragments by pcr amplification with specific primers (f: ′-ttctgccgcctgcttcaaaccg- ', r: ′-ctcggatatccctaatcc- '; f: ′-ttggttgaactccggaac- ', r: ′-cagaatgggcagacattacgaatgc- '). a backbone vector pt , which contains the t rna polymerase promoter, encephalomyocarditis (emc) virus internal ribosome entry site (ires), and the t terminator sequences, was used to construct plasmids encoding mv-hu n, p, and l genes. the open reading frames (orfs) of the mv hu n and p genes were amplified from peasy-n and peasy-p using primer pairs mv-cds-n (+)/(-) and mv-cds-p (+)/(-), respectively, whereas the orf of the mv hu l gene was amplified from peasy-l and peasy-l using two primer pairs, mv-cds-l (+)/(-) and mv-cds-l (+)/(-). the mv n, p, and l genes were inserted into the pt vector between the ires and polya table replication of rmv-hu mutants in cotton rats. % infected animals viral titer (log pfu/g) b,c rmv-hu . ± . a rmv-hu -g a . ± . b rmv-hu -g a . b dmem nd nd: not detected. a each cotton rat was inoculated intranasally with . × pfu of rmv-hu or rmv-hu mutants in a volume of µl. at day post-infection, the cotton rats were sacrificed, and lungs were collected for both virus titration and rt-pcr. b the viral titer was determined by plaque assay. c five cotton rats were tested in each group. values within a column followed by different capital letters (a and b) are significantly different. fig. . neutralizing antibody titers produced by cotton rats after inoculation with recombinant mv. cotton rats were intranasally inoculated with . × pfu of rmv-hu or rmv-hu mutants in . ml of opti-mem medium. (a) weekly blood samples were collected from each cotton rat by facial vein retro-orbital bleeding, and serum was tested for neutralizing antibodies by a plaque-reduction neutralization assay. (b) recombinant mvs carrying mutations in the sam binding site elicited significantly higher levels (p < . ) of neutralizing antibodies at weeks post-inoculation. * =p < . ; * *=p < . ; *** =p < . . sequences using a "seamless" cloning strategy, resulted in the construction of pt -hu -n, pt -hu -p, and pt -hu -l, respectively. the primer sequences used in the pcr assays are available upon request. to recover recombinant mv, bhk-sr -t cells were grown overnight in six-well plates to approximately % confluence, and were transfected with µg of pyes-mv(+), . µg of pt -hu -n, . µg of pt -hu -p, and . µg of pt -hu -l using previously described procedure (carsillo et al., ; kovacs et al., ; singh and billeter, ) . at h post-transfection, cell monolayers were trypsinized and directly transferred onto vero cell monolayers (p ) at % confluence and co-cultured at °c for - days. cells were subjected to three freeze-thaw cycles when extensive cpe (mv-induced syncytia) was observed. after a brief centrifugation, supernatants (p ) were harvested and used for further passages on confluent vero cell monolayers. at p or p , the recovered viruses were plaque purified and sequenced. vero cells grown in -well tissue culture plates were infected with rmv or rmv-mutant. after h incubation, the cells were washed two times with pbs before cultivating them in dmem containing % fbs. at or h postinfection, the cells were fixed with % paraformaldehyde in pbs for min at rt, permeabilized with . % triton x- (merck millipore) in pbs for min at rt, and blocked with % bsa in pbs containing . % tween for h at rt. the cells were stained with mouse anti-measles virus n antibody (ab , abcam) for h at rt. after washing with pbs, alexa fluor ® donkey anti-mouse igg (h+l) (a , invitrogen) were added and incubated for h at rt. then, the cells were stained with , -diamidino- -phenylindole (dapi) for min at rt. images were obtained using a zeiss clsm confocal laser scanning microscope and zen software. amino acids (g , g , and g ) in the sam binding site in the hu l protein were mutated to alanine individually (ggt to gct at aa posi tion , gga to gca at aa position , and ggt to gct at aa position ; the mutated nucleotides were underlined) in an infectious cdna clone of mv-hu [pyes-mv(+)] using a q ® sitedirected mutagenesis kit (neb) according to the manufacturer's instructions. the resultant plasmids were named pyes-mv(+)-g a, pyes-mv(+)-g a, and pyes-mv(+)-g a. mutations were confirmed by dna sequencing. the titers of rmv-hu viruses were determined by plaque assay in vero cells. briefly, vero cells were seeded in six-well plates at a density of × cells per well, incubated for h, and the medium was removed prior to infection of cell monolayers with serial dilutions of rmv-hu . after h of adsorption with constant shaking, the medium was removed and cell monolayers were covered with . ml of eagle's minimal essential media (mem) containing % agarose, . % sodium bicarbonate(nahco ), % fbs, nm hepes, mm l-glutamine, and mg/ml of streptomycin. at dpi, cells were fixed in % (vol/vol) paraformaldehyde for h, and the plaques were visualized by staining with . % (wt/vol) crystal violet. confluent vero cells in six-well plates were infected with rmv-hu viruses at a multiplicity of infection (moi) of . . after h incubation, the inoculum was removed and the cells were washed three times with pbs. fresh maintenance media (dmem supplemented with % fbs) was added, and the infected cells were incubated at °c. at different time points post-infection, the cells were harvested by three freeze-thaw cycles, and the supernatant collected by centrifugation at ×g in an allegra r centrifuge (beckman coulter) for min. virus titers were determined by plaque assay in vero cells. confluent vero cells in t flasks were infected with each rmv-hu mutant at an moi of . , and cell culture supernatants were collected after appearance of cpe and used to infect new confluent vero cells in fresh t flasks. each mutant was serially passaged times in vero cells. viral rna was extracted from cell culture supernatant harvested from each passage. the cr vi of the l gene was amplified by rt-pcr and sequenced. additionally, the entire genome of each recombinant virus was amplified by rt-pcr and sequenced at passage . all the plasmids, viral stocks, and virus isolates from the lungs of cotton rats were sequenced. viral rna was isolated using an rneasy mini-kit (qiagen) according to the manufacturer's instructions. viral rna was treated with dnase i to eliminate possible contamination from original transfecting plasmid dna, and no-rt pcr controls were carried out to confirm complete digestion of plasmid dna. a . kb dna fragment of the h protein gene was amplified by a one-step rt-pcr kit (qiagen) using primers rmv-h- -forward ( 'gttcagggatggacctatac- ') and rmv-l- -reverse ( 'ggtgtgtgtctcctcctat- '). pcr products were sequenced to ensure that the isolated virus was rescued from pyes-mv(+) and not from the contamination of the wild type mv-hu grown in our laboratory. a . kb dna fragment spanning cr vi of the mv l-protein was amplified by a one-step rt-pcr kit (qiagen) using primers rmv-l- -forward ( '-gaccggtagagaaatgtgcag- ') and rmv-l- -reverse ( '-gcttaatggataggatgtgac- '). pcr products were sequenced to confirm that each recombinant virus contained the desired mutation. the rmv-hu stocks for use in animal experiments were grown in vero cells and purified by ultracentrifugation. twenty t flasks with confluent vero cells were infected with each rmv at moi of . . after h of adsorption with constant shaking, ml of dmem (supplemented with % fbs) was added to each flask and incubated at °c until extensive cpe was observed. the cells were harvested using a cellscraper, and suspensions were clarified by centrifugation at ×g for table immunogenicity of mtase-defective rmv-hu mutants in cotton rats a . inoculum a % infected animals viral titer (log pfu/g) b rmv-hu nd rmv-hu -g a nd rmv-hu -g a nd dmem . ± . nd: not detected. a cotton rats were intranasally inoculated with . × pfu of rmv-hu or mutants. at days post-infection, rats were challenged with × pfu of rmv-hu . at day post-challenge, cotton rats were sacrificed and lungs were collected for both virus titration and rt-pcr. b the viral titer was determined by plaque assay. min at °c in an allegra r centrifuge (beckman coulter). the cell pellets were resuspended in ml of dmem and subjected to three freeze-thaw cycles, clarified by low-speed centrifugation, and the supernatants were combined. the virus was pelleted by ultracentrifugation at , ×g in a beckman ty . rotor for h, and resuspended in . ml of dmem, aliquoted, and stored. viral titer was determined by plaque assay. twenty - week-old female specific-pathogen-free (spf) cotton rats (envigo, indianapolis, in) were randomly divided into four groups ( cotton rats per group), and housed within the ular facilities at the ohio state university according to iacuc policies and guidelines (animal protocol no. a ). each inoculated group was separately housed in rodent cages under biosafety level conditions; rats were anesthetized with isoflurane before virus inoculation. cotton rats in groups - were inoculated with parental rmv-hu , rmv-hu -g a, and rmv-hu -g a, respectively. cotton rats in group were mock-infected with dmem, and served as uninfected controls. each cotton rat was inoculated intranasally with × pfu of virus in a volume of µl. at dpi, cotton rats were sacrificed and lungs were collected for virus titration and rt-pcr. for the immunogenicity study, twenty five - week-old cotton rats (envigo) were randomly divided into five groups ( cotton rats per group). cotton rats in groups were mock-infected with dmem and served as uninfected unchallenged control. cotton rats in groups , and were intranasally inoculated with . × pfu of rmv-hu , rmv-hu -g a, and rmv-hu -g a, respectively. cotton rats in groups were mock-infected with dmem and served as uninfected challenged control. after immunization, the cotton rats were evaluated daily for mortality, and blood samples were collected from each cotton rat weekly by facial vein retro-orbital bleeding, and the serum was used for detection of neutralizing antibodies. at weeks post-immunization, the cotton rats in groups - were challenged with . × pfu of parental rmv-hu via intranasal route, and evaluated twice daily for the presence of any clinical symptoms. at days post-challenge, all cotton rats were euthanized by co asphyxiation, and their lungs were collected for virus titration. the immunogenicity of rmv-hu mutants was assessed based on their ability to trigger neutralizing antibodies and the ability to protect mv replication in lungs. serum neutralization of virus was performed using an endpoint dilution plaque reduction assay; and (ii) quantification of lung viral titers was done by plaque assay. mv-specific neutralizing antibody was determined using an endpoint dilution plaque reduction assay. briefly, cotton rat sera were collected weekly until challenge. the serum samples were heat inactivated at °c for min. two-fold dilutions of the serum samples were mixed with an equal volume of dmem containing approximately pfu/well rmv-hu in a -well plate, and the plate was incubated at room temperature for h with constant rotation. the mixtures were then transferred to confluent vero cells in a -well plate in triplicate. after h of incubation at °c, the virus-serum mixtures were removed and the cell monolayers were covered with . ml of eagle's minimal essential media (mem) containing % agarose, . % sodium bicarbonate(nahco ), % fbs, nm hepes, mm l-glutamine, and mg/ml of streptomycin. then, the cells were incubated for another days before virus plaque titration as described above. the plaques were counted, and % plaque reduction titers were calculated as the mv-specific neutralizing antibody titers. statistical analysis was performed by one-way multiple comparisons; two-way multiple comparisons (anova) using prism statistical analysis software (version . ). p value of < . was considered statistically significant. measles and measles vaccination: a review generation of bovine respiratory syncytial virus (brsv) from cdna: brsv ns is not essential for virus replication in tissue culture, and the human rsv leader region acts as a functional brsv genome promoter recovery of infectious respiratory syncytial virus expressing an additional, foreign gene cytokine imbalance after measles virus infection has no correlation with immune suppression rescue of mumps virus from cdna measles vaccination: new strategies and formulations observation of measles virus cell-to-cell spread in astrocytoma cells by using a green fluorescent protein-expressing recombinant virus viral rna-polymerases -a predicted '-o-ribose methyltransferase domain shared by all mononegavirales viral and cellular mrna capping: past and prospects the measles epidemic trend over the past years in a central district a highly recombinogenic system for the recovery of infectious sendai paramyxovirus from cdna: generation of a novel copy-back nondefective interfering virus establishment of a rescue system for canine distemper virus measles. history and basic biology recombinant human respiratory syncytial virus (rsv) from cdna and construction of subgroup a and b chimeric rsv enhanced genetic rescue of negative-strand rna viruses: use of an mva-t rna polyrnerase vector and dna replication inhibitors recombinant vesicular stomatitis viruses from dna a conserved motif in region v of the large polymerase proteins of nonsegmented negative-sense rna viruses that is essential for mrna capping a unique strategy for mrna cap methylation used y. wang et al by vesicular stomatitis virus measles transmission among adults with spread to children during an outbreak: implications for measles elimination in china mrna cap methylation influences pathogenesis of vesicular stomatitis virus in vivo nucleotide sequence analysis of the large (l) genes of phocine distemper virus and canine distemper virus (corrected sequence) rescue system for measles virus from cloned cdna driven by vaccinia virus lister vaccine strain a decade after the generation of a negative-sense rna virus from cloned cdna -what have we learned? unconventional mechanism of mrna capping by the rna-dependent rna polymerase of vesicular stomatitis virus sendai virus rna-dependent rna polymerase l protein catalyzes cap methylation of virus-specific mrna enhanced measles virus cdna rescue and gene expression after heat shock sequence comparison of five polymerases (l proteins) of unsegmented negative-strand rna viruses: theoretical assignment of functional domains rescue of measles viruses from cloned dna ribose '-o methylation of the vesicular stomatitis virus mrna cap precedes and facilitates subsequent guanine-n- methylation by the large polymerase protein universal catalytic domain structure of adomet-dependent methyltransferases measles vaccine adverse events reported in the mass vaccination campaign of sichuan province, china from rescue of synthetic measles-virus minireplicons -measles genomic termini direct efficient expression and propagation of a reporter gene a recombinant measles virus expressing biologically active human interleukin- methyltransferase-defective avian metapneumovirus vaccines provide complete protection against challenge with the homologous colorado strain and the heterologous minnesota strain live attenuated measles vaccine as a potential multivalent pediatric vaccination vector critical role of k and k in the large protein of rabies virus in viral pathogenicity and immune evasion transcription and replication of nonsegmented negative-strand rna viruses rescue of wild-type mumps virus from a strain associated with recent outbreaks helps to define the role of the sh orf in the pathogenesis of mumps virus rational design of human metapneumovirus live attenuated vaccine candidates by inhibiting viral mrna cap methyltransferase genetic characterization of chinese measles vaccines by analysis of complete genomic sequences measles outbreak among previously immunized adult healthcare workers, china, . can the authors declare that they have no competing interests. key: cord- -fad yyqx authors: felgenhauer, ulrike; schoen, andreas; gad, hans henrik; hartmann, rune; schaubmar, andreas r.; failing, klaus; drosten, christian; weber, friedemann title: inhibition of sars–cov- by type i and type iii interferons date: - - journal: j biol chem doi: . /jbc.ac . sha: doc_id: cord_uid: fad yyqx the recently emerged severe acute respiratory syndrome coronavirus- (sars–cov- ) is the causative agent of the devastating covid- lung disease pandemic. here, we tested the inhibitory activities of the antiviral interferons of type i (ifn-α) and type iii (ifn-λ) against sars–cov- and compared them with those against sars–cov- , which emerged in . using two mammalian epithelial cell lines (human calu- and simian vero e ), we found that both ifns dose-dependently inhibit sars–cov- . in contrast, sars–cov- was restricted only by ifn-α in these cell lines. sars–cov- generally exhibited a broader ifn sensitivity than sars–cov- . moreover, ruxolitinib, an inhibitor of ifn-triggered janus kinase/signal transducer and activator of transcription signaling, boosted sars–cov- replication in the ifn-competent calu- cells. we conclude that sars–cov- is sensitive to exogenously added ifns. this finding suggests that type i and especially the less adverse effect–prone type iii ifn are good candidates for the management of covid- . we tested the effect of type i ifn against sars-cov- compared with the sars-cov- from . two different cell lines were employed, namely the human bronchial epithelial calu- and the primate kidney epithelial vero e . the cells were first treated for h with , , or units/ml of recombinant human ifn-a(b/d) and then infected with the viruses at a multiplicity of infection (moi) of . plaque forming units (pfu)/cell to obtain multistep growth. virus titers in supernatants were determined h later, when titers are reaching a plateau (see below). the data of three biological replicates are shown in fig. . because several titers were below the detection limit of our plaque assay, a rank correlation test (spearman's exact rank correlation test) was used for statistical doseresponse correlation analysis. for sars-cov- (dark gray bars), statistically significant negative correlation coefficients (cc) were obtained for both cell lines, indicating that viral replication is increasingly inhibited by ifn-a. for sars-cov- (light gray bars), titers were also affected. however, at least in vero e cells, the reduction of sars-cov- appears to be weaker than the reduction of sars-cov- (fig. b) . observations were similar when the input moi was reduced to . (fig. s ), except that titers of sars-cov- in calu- cells were already very low in the absence of any ifn-a, resulting in a nonsignificant effect of additional ifn. these data may suggest that the potency of ifn to reduce viral titers may be stronger and more consistent against sars-cov- than against sars-cov- . to further investigate the potential differences between the viruses, we repeated the experiment three times more with the intermediate dose of units/ml and analyzed the data statistically after pooling them with the previous three replicates. two-way anova was used to simultaneously evaluate the influence of both ifn-a and virus species on virus reduction. this analysis (fig. , a and b) showed again that (i) both viruses are reduced by ifn (comparison of versus units/ml ifna, p(ifn)) and (ii) there are differences between the sars-cov species (comparison of the virus experiments, p(virus)). moreover, the "interaction" p value showed that, at least in vero cells, the degree of ifn sensitivity depends on the virus species, again indicating that sars-cov- is more ifn-sensitive than sars-cov- . the predominant antiviral cytokine ( , ) . although the ifn induction as well as signaling and up-regulation of ifn-stimulated genes (isgs) are very similar, type iii ifns engage a different receptor that is restricted to epithelial cells, and generate a weaker but longer-lasting antiviral response ( , ) . ifn-l was previously shown to have activity against coronaviruses ( , , ) and proposed as potential covid- treatment ( ) . hence, we compared the sensitivity of the two sars-coronaviruses also to recombinant human ifn-l. as shown in fig. a , pretreatment with or ng/ml ifn-l exhibited only in vero e cells a dose-dependent inhibitory effect on sars-cov- . for sars-cov- , by contrast, no significant inhibition was noted in any of the cell lines. to further investigate the difference between the viruses, we repeated the ifn-l experiment three times more with the intermediate dose of ng/ml and analyzed the data after pooling with the previous ng/ml ifnl experiment (fig. b ). conventional statistical analysis (onetailed student's t test, because none of the values was below the detection limit) again revealed a significant impact of ifn-l on sars-cov- and the lack of an effect for sars-cov- . our data thus show that ifn-l can inhibit sars-cov- but not sars-cov- . a recent study on the host cell interactome of sars-cov- identified a number of human proteins for which food and drug administration-approved drugs are available ( ) . ruxolitinib, a compound known to target the type i and type iii ifntriggered jak/stat signaling pathway ( ) , was among the proposed inhibitors of virus-host cell interactions ( ) . because virus inhibition by an ifn inhibitor seems counterintuitive, we aimed to clarify the influence of this compound on sars-cov- replication. cells were pretreated with mm ruxolitinib for h and infected at the two different mois, and titers were measured or h later. as shown in fig. , with this setting titers in nontreated controls are already reaching a plateau at the -h time point. in calu- cells, ruxolitinib had a ( ) . our data thus indicate that (i) if anything, ruxolitinib is an enhancer rather than an inhibitor of sars-cov- multiplication, and (ii) the boosting effect is most likely due to inhibition of the antiviral jak/ stat signaling pathway, because it is not present in the ifn induction-deficient vero e cells. our observations so far suggest that sars-cov- is consistently more sensitive to ifns than sars-cov- . moreover, type i ifn seems to have a more profound effect than type iii ifn. to see whether principal differences in signaling or subsequent gene expression could account for these phenomena, we tested the ability of the cell lines to respond to the ifns. the immunoblot analysis (fig. ) shows that calu- cells have a very similar reaction to both types of ifn concerning phosphorylation of stat and stat and expression of the ifn-stimulated mxa and isg . vero e cells also responded to ifn-l as expected ( ) , but the isg response was lower than to ifn-a. moreover, in nontreated calu- cells, there was already a background isg expression, which was not observed in vero cells. ruxolitinib was in principle able to influence these isg responses, as expected, but it was more potent against ifn-l than against ifn-a, and its effects on ifn-stimulated genes were more evident in the vero e compared with the calu- (fig. ) . thus, both cell lines are capable to respond to the different types of ifn, but ifn-l was less potent, which is in agreement with our observations on sars-cov sensitivity, as well as with previous studies ( , ) . the recently emerged sars-cov- is responsible for major health crises all over the world. here, we show that type i and type iii ifns are able to inhibit sars-cov- replication, with effects that in our hands were consistently more profound than against the sars-cov- from . it should be noted however, that the differences between the viruses could be due to the cell types used or due to the observed differences in virus replication (which could result in higher production of ifn antagonists). thus, the question whether sars-cov- is intrinsically more sensitive to ifns remains to be solved. pegylated ifn-a was the standard of care against chronic infection with hepatitis c virus until the recent introduction of other, directly acting antiviral drugs ( ) . although associated with some side effects, ifn-a is well-characterized, has been used to treat millions of patients, is considered safe, and is available figure . sensitivity of sars-cov- and sars-cov- to type iii ifn. a, experiments were performed as described for fig. , except that recombinant human ifn-l was used. log-transformed titers of each virus dose-response experiment with concentrations of and ng/ml ifn-l were analyzed by spearman's exact rank correlation test. ccs and exact one-sided p values are provided. b, three additional biological replicates of the ng/ml ifn-l were performed, and the resulting titer data were pooled with the ng/ml ifn-l data from a. log-transformed titers were analyzed by unpaired one-tailed student's t test. n.s., nonsignificant. immediately. ifn-l has undergone phase i and ii clinical trials with hepatitis c virus ( ) . it exhibited excellent tolerance as well as efficacy, but the phase iii trials were abandoned because of the availability of effective direct antivirals. ifn-l holds promise as having fewer side effects because of its restriction to mucosal tissue and the less sudden but more prolonged antiviral response it triggers ( , ) . in line with our results, a series of preprints show that also others found type i and type iii ifns to be effective against sars-cov- replication in cell culture ( ) ( ) ( ) . also in earlier in vivo studies with sars-cov- , both type i and type iii ifns were shown to be important for the control of infection or the associated disease ( - ). clinical data on the usage of type i ifn against sars-cov- or the related mers-cov, however, are limited, are not always conclusive, or did not show a clear benefit ( ) ( ) ( ) ( ) ( ) . thus, type iii ifn-l rather than the side effect-prone type i ifns ( ) might be considered for clinical testing against sars-cov- . ruxolitinib was proposed as a potential treatment against sars-cov- ( , ) , and a small clinical trial is underway (clinicaltrials.gov, nct ), although case reports were discouraging ( ) . the replication boost obtained with ruxolitinib on the ifn-competent calu- cells indicates that ruxolitinib is not at all inhibiting sars-cov- replication. thus, drugs that interfere with viral host interactors may not necessarily be antiviral but rather boost the infection. calu- and vero e cells were cultivated in dulbecco's modified eagle's medium supplemented with % fetal bovine serum (thermo fisher scientific) in a % co atmosphere at °c. sars-cov- (strain sars-cov- /münchen- . / / , p. ) ( ) and sars-cov- (strain sars-fra , p. ) ( ) were grown on vero e cells and purified via vivaspin columns (sartorius stedim biotech). the viruses were titrated on vero e cells. infection experiments were done under biosafety level conditions with enhanced respiratory personal protection equipment. of note, all cells were tested mycoplasma-negative. the cells were pretreated for h with the indicated amounts of pan-species ifn-a(b/d) (pbl assay science) ( ), purified recombinant ifn-l ( , ) , or with mm ruxolitinib (selleckchem). infections were performed at a moi of . and . . at the indicated times postinfection, cell supernatants were collected and titrated by plaque assay on vero e cells. the cells were treated for h with the indicated amounts of ifns or ruxolitinib (added h before ifn) and lysed in t-per protein extraction reagent (thermo fisher) supplemented containing protease inhibitor mixture (c mplete, roche), phosphatase inhibitor mixture set ii (calbiochem), and sample buffer ( . mm tris-hcl, ph . , . % glycerol, . % sds, . mm bromphenol blue). protein samples were run on % acrylamide gels and transferred to polyvinylidene fluoride membranes (millipore) via semidry blotting. after blocking in tbs with % bsa (for detection of phospho-stats, mxa, and total stat ) or milk powder (all other detections), primary antibody staining was performed overnight at °c. the membranes were washed in tbs with . % tween , stained with secondary antibodies for min, and washed again in tbs with . % tween and once in tbs. finally, the membranes were developed with a supersignal west femto kit (pierce), and the bands were visualized using a chemidoc imaging system (bio-rad). the the statistical analysis of the data were done by means of the statistical program packages bmdp ( ) and statxact ® (version . . ). for the statistical testing of the dose-response effect of ifn (type i and iii) against sars-coronaviruses, the typical regression procedures were not applicable because of several values below the detection limit and some ties in the data. instead of this, the nonparametric spearman rank cc was used in the exact version (software statxact). because the scientific question was clearly one-sided formed (only pfu reduction under application of ifn), one-sided p values were given. if only two ifn concentrations were to compare with no data below the detection limit, then the t test for independent sam-ples was used (program bmdp d). for testing the effect of ifn and virus type simultaneously, the two-way anova (program bmdp d) was applied especially considering a possible interaction between the two tested factors. in the parametric statistical analyses as well as the graphical representations, the response variable pfu was logarithmically transformed because of its right skewed statistical distribution. in all cases a statistical significance level of a = . was applied. all data presented and discussed are contained within the article. author contributions-u. f., a. s., r. h., a. r. s., k. f., c. d., and f. w. conceptualization; u. f., c. d., and f. w. validation; u. f., a. s., a. r. s., and k. f. investigation; u. f. and f. w. visualization; coronaviridae study group of the international committee on taxonomy of viruses ( ) the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- genome composition and divergence of the novel coronavirus ( -ncov) originating in china shared and distinct functions of type i and type iii interferons guarding the frontiers: the biology of type iii interferons interferon-l orchestrates innate and adaptive mucosal immune responses interferon lambda: disease impact and therapeutic potential type i interferon in chronic virus infection and cancer tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures treatment of sars with human interferons inhibition of novel b coronavirus replication by a combination of interferon-a b and ribavirin efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential interaction of sars and mers coronaviruses with the antiviral interferon response the antiviral effect of interferon-beta against sars-coronavirus is not mediated by mxa protein severe acute respiratory syndrome related coronavirus is inhibited by interferon-alpha human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus coronavirus spike protein and tropism changes differential induction of interferon stimulated genes between type i and type iii interferons is independent of interferon receptor abundance interferon lambda signals via the ifnl receptor to regulate antiviral activity against hcv and coronaviruses lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections covid- and emerging viral infections: the case for interferon lambda a sars-cov- protein interaction map reveals targets for drug repurposing comprehensive analysis of kinase inhibitor selectivity alpha/beta interferon (ifn-alpha/ beta)-independent induction of ifn-lambda (interleukin- ) in response to hantaan virus infection peginterferon alfa- a plus ribavirin for chronic hepatitis c virus infection a randomized phase b study of peginterferon lambda- a for the treatment of chronic hcv infection sars-cov- is sensitive to type i interferon pretreatment potent antiviral activities of type i interferons to sars-cov- infection ) critical role of type iii interferon in controlling sars-cov- infection, replication and spread in primary human intestinal epithelial cells. biorxiv crossref sars-cov pathogenesis is regulated by a stat dependent but a type i, ii and iii interferon receptor independent mechanism pegylated interferon-alpha protects type pneumocytes against sars coronavirus infection in macaques combined action of type i and type iii interferon restricts initial replication of severe acute respiratory syndrome coronavirus in the lung but fails to inhibit systemic virus spread interferon-lambda contributes to innate immunity of mice against influenza a virus but not against hepatotropic viruses ribavirin and interferon therapy for critically ill patients with middle east respiratory syndrome: a multicenter observational study interferon alfacon- plus corticosteroids in severe acute respiratory syndrome: a preliminary study ribavirin and interferon alfa- a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study sensitivity of sars/ mers cov to interferons and other drugs based on achievable serum concentrations in humans sars: systematic review of treatment effects disease-promoting effects of type i interferons in viral, bacterial, and coinfections covid- : combining antiviral and anti-inflammatory treatments side effects of ruxolitinib in patients with sars-cov- infection: two case reports transmission of -ncov infection from an asymptomatic contact in germany identification of a novel coronavirus in patients with severe acute respiratory syndrome a recombinant human interferon-alpha b/d hybrid with a broad host-range the influence of the rs single nucleotide polymorphism on ifn-l activity and secretion bmdp statistical software manual key: cord- - vim wno authors: zandi, keivan; teoh, boon-teong; sam, sing-sin; wong, pooi-fong; mustafa, mohd rais; abubakar, sazaly title: antiviral activity of four types of bioflavonoid against dengue virus type- date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: vim wno background: dengue is a major mosquito-borne disease currently with no effective antiviral or vaccine available. effort to find antivirals for it has focused on bioflavonoids, a plant-derived polyphenolic compounds with many potential health benefits. in the present study, antiviral activity of four types of bioflavonoid against dengue virus type - (denv- ) in vero cell was evaluated. anti-dengue activity of these compounds was determined at different stages of denv- infection and replication cycle. denv replication was measured by foci forming unit reduction assay (ffura) and quantitative rt-pcr. selectivity index value (si) was determined as the ratio of cytotoxic concentration (cc( )) to inhibitory concentration (ic( )) for each compound. results: the half maximal inhibitory concentration (ic( )) of quercetin against dengue virus was . μg ml(- )when it was used after virus adsorption to the cells. the ic( )decreased to . μg ml(- )when the cells were treated continuously for h before virus infection and up to days post-infection. the si values for quercetin were . and . μg ml(- ), respectively, the highest compared to all bioflavonoids studied. naringin only exhibited anti-adsorption effects against denv- with ic( )= . μg ml(- )and its related si was . . daidzein showed a weak anti-dengue activity with ic( )= . μg ml(- )when the denv- infected cells were treated after virus adsorption. the si value for this compound was . . hesperetin did not exhibit any antiviral activity against denv- . the findings obtained from foci forming unit reduction assay (ffura) were corroborated by findings of the qrt-pcr assays. quercetin and daidzein ( μg ml(- )) reduced denv- rna levels by % and %, respectively. there was no significant inhibition of denv- rna levels with naringin and hesperetin. conclusion: results from the study suggest that only quercetin demonstrated significant anti-denv- inhibitory activities. other bioflavonoids, including daidzein, naringin and hesperetin showed minimal to no significant inhibition of denv- virus replication. these findings, together with those previously reported suggest that select group of bioflavonoids including quercetin and fisetin, exhibited significant inhibitory activities against dengue virus. this group of flavonoids, flavonol, could be investigated further to discover the common mechanisms of inhibition of dengue virus replication. dengue virus (denv) is a member of the genus flavivirus of the flaviviridae family. it is a significant human pathogen which causes a wide spectrum of clinical illnesses ranging from a silent or mild febrile infection, self-limited dengue fever (df) to the severe dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss). there are four dengue virus genotypes, denv- , denv- , denv- and denv- which are transmitted to humans mainly by two species of mosquitoes, aedes agypti and aedes albopictus [ ] . all four denv can cause dengue. to date there are no effective vaccine or antiviral treatment for dengue. dengue patients are usually supportively-treated until they recover without any specific treatment measures. several studies have shown that the level of viremia correlates with the severity of disease with high viremia often seen in severe dengue. hence, antivirals that can reduce the level of viremia or the viremic phase could possibly reduce the severity of dengue. plants and plant's derived compounds remain an important source for the discovery and the development of new antiviral drugs because of their expected low side effects and their high accessibility in the nature [ ] [ ] [ ] . there have been numerous reports on the antiviral activity of various phytochemicals against dengue viruses and these include various flavonoids [ ] [ ] [ ] [ ] . flavonoids are basically low molecular weight phenolic compounds found widely in different kinds of plants. different types of flavonoids can be found in fruits, roots, nuts, seeds, bark, steams and flowers of plants. these include quercetin which can be found in some foods and fruits such as green and black tea, apple, onion, citrus, tomato and some other plants [ , ] . antiviral activities of various other flavonoids have also been reported against some viruses including human cytomegalovirus (hcmv), hsv- , hsv- and some types of human adenoviruses [ ] [ ] [ ] . in the present study, we are interested to examine the anti-dengue virus properties of quercetin, hesperetin, naringin and daidzein. hesperetin is a flavonone and its glycoside form, hesperidin is water soluble and it could be found in various citrus fruits. after ingestion of hesperidin, its sugar moiety is released from the backbone of this compound and the aglycone form known as hesperetin can be released. in vitro antiviral activities of hesperetin have been reported against some rna viruses [ ] [ ] [ ] . naringin on the other hand, is a flavonone glycoside found abundantly in grapefruit juice. antiviral activity of naringin were reported against hsv- and hsv- but this finding remains controversial [ , ] . daidzein is an isoflavone found in soybeans and its antiviral activity against influenza viruses has been reported [ ] . currently, there is no published data on the possible anti-dengue virus activities of quercetin, hesperetin, naringin and daidzein. therefore, in this study we evaluated these compounds activities on denv- (ngc strain) replication in cell culture system. the effects of each compound were evaluated against different stages of dengue virus replication including virus adsorption, intracellular replication and direct virucidal activities. four different types of bioflavonoid, quercetin, naringin, hesperetin (sigma-aldrich, st. louis, mo, usa) and daidzein (indofine chemical co. inc., hillsborough, nj, usa) were evaluated for their potential activities against dengue virus replication. dimethyl sulfoxide (dmso) (sigma-aldrich, st. louis, mo, usa) was used to dissolve the lyophilized form of compounds and prepared stock solutions ( mg ml - ) were stored at - c. stock solution was diluted using cell culture medium and sterilized by a syringe filter with . μm pore size (millipore, ma, usa) right before each experiment. c / mosquito cell line derived from aedes albopictus and vero (african green monkey kidney) cell line were used in this study. both cell lines were maintained and propagated in eagle's minimum essential medium (emem) (gibco, ny, usa) containing % fetal bovine serum (gibco, ny, usa). cultured c / and vero cells were incubated at c and c, respectively in % co humidified chamber. at the time of virus propagation, serum concentration was reduced to %. dengue virus type- (denv- ) new guinea c strain (ngc) was propagated using c / cell line and harvested after cpe presentation on day seven post-infection. after titration, viral stock was stored at - c. cell lines and virus were provided by virology laboratory of the tropical infectious disease research and education center, faculty of medicine, university of malaya (kuala lumpur, malaysia). mtt assay was performed following the manufacturer's instructions (promega, wi, usa). briefly, confluent vero cells in -well cell culture microplates were treated with different concentrations of each compound in triplicate. the treated cells were incubated for four days at c followed by the addition of μl of mtt solution to each well. the microplate was incubated at c for h. then, μl of the solubilisation/stopping solution was added to each well. the optical density (od) of wells was measured at using -well plate reader (tecan, mannendorf, switzerland). dose-response curves were plotted using graph pad prism (graph pad software inc., san diego, ca). in order to determine the prophylactic anti-dengue activity of compounds, different concentrations of compounds were added to the vero monolayer cells in triplicate at different times, h before virus infection. after h of pre-infection treatment, the cells were washed twice with sterile pbs and then ffu of denv- was inoculated to the cells and incubated at c for h. to determine the effects of continuous treatment, different concentrations of each compound were added to the vero cells, h pre-infection and continuously for days post-infection. in a separate experiment, antiviral activity of compounds against intracellular replication of denv- was performed by inoculation of ffu of virus to each well in triplicate. after adsorption of virus to the cells for h at c, the cells were washed with pbs to eliminate the unabsorbed viruses. then, different concentrations of each compound were added to the cells, followed by days of incubation at c. denv rna was then quantified using quantitative rt-pcr. in another experiment, vero cells at % confluency were infected with ffu of denv- in the presence or absence of different concentrations of each compound. the microplate was kept at c for h for virus adsorption. then the cells were washed two times by sterile pbs and incubated at c for four days. direct virucidal effects of the bioflavonoids were investigated by incubating denv- suspension containing ffu with an equal volume of the different concentrations of each compound for h at c. then, vero cells were infected with the treated viral suspension in triplicate. after h adsorption at c, cells were washed twice with pbs in order to remove unabsorbed viruses. then the microplate was incubated at c for days. antiviral activities of the tested compounds were evaluated by measuring the reduction in number of viral foci. briefly, confluent monolayers of vero cells were prepared in wells cell culture microplate. then the infected cells treated using different procedures described above were overlaid with . % of carboxy methyl cellulose (cmc) (sigma-aldrich, st. louis, mo, usa) containing emem. viral foci were visualized using peroxidase-based foci staining assay four days post infection [ ] . the numbers of denv- foci were counted using a stereomicroscope and the titer of virus was expressed as foci-forming-unit (ffu). the percentage of viral foci reduction (rf %) compared with controls was calculated as follows: rf (%) = (c-t) × /c. where, c is the mean of the number of foci for negative control well (without compound) and t is the mean of the number of foci in treated wells. reduction in the number of viral foci was further verified using quantitative rt-pcr (qrt-pcr). quantitative rt-pcr was performed to determine the effects of bioflavonoids on denv replication by quantifying denv- genomic rna copies based on a method described previously with some modifications [ ] . briefly, intracellular and extracellular denv- rnas were harvested from the denv-infected vero cells. viral rna was extracted using two types of rna extraction kits (qiaamp viral rna mini kit and rneasy mini kit) (qiagen, hilden, germany). quantitative rt-pcr assay was performed by adding μl of extracted denv rna to the sensimix sybr green reagent (quantace, watford, united kingdom) which contained . μl ddh o, μl x sensimix one-step, . μl x sybr green solution, units of rnase inhibitor, pmol of forward (dnf) and also reverse (d r) primers [ ] . all samples were assayed in triplicate. the amplifications were performed using the dna engine opticon system (mj research/bio-rad, hercules, ca) with the following thermal conditions: reverse transcription at °c for min, initial denaturation at °c for min, followed by cycles of °c for sec, °c for sec and °c for sec. melting curve analysis was subsequently performed at temperature from °c to °c to verify the assay specificity. for absolute quantitation of the viral rna, a standard curve was established with a serially diluted rna extracted from denv- stock with known titer. graph pad prism for windows, version (graph pad software inc., san diego, ca, ) was used to determine the cytotoxic concentration (cc ) and inhibitory concentration (ic ) values of bioflavonoids. selectivity index value (si) was determined as the ratio of cc to ic for each compound. mtt assay was used to determine cytotoxicity of each bioflavonoid on vero cells and the cc value of each compound was calculated (table and figure ). vero cells were treated by bioflavonoids for days which was the same duration used for antiviral activity assay. results showed that hesperetin with cc = . ± . μg ml - is the most cytotoxic compound for vero cells compared to the other tested compounds. quercetin and daidzein showed lower toxicity against vero cells at cc . ± . and . ± . μg ml - , respectively. meanwhile, naringin with cc = . ± . μg ml - showed the least cytotoxic effects against vero cells. cells treated with vehicle control, % dmso did not show any cytotoxicity against vero cells. results of vero cells pre-treatment with the compounds showed that μg ml - of quercetin could decrease the number of denv- foci by % ± . when compared to the non-treated cells. however, there was no prophylactic activity against denv- from other compounds (data not shown). in post-adsorption assay, quercetin exhibited the most significant antiviral activity against denv- amongst the bioflavonoids tested with ic = . μg ml - (figure a) . the si value for quercetin in post-adsorption assay was relatively high at . . it was also demonstrated that the level of denv- specific rna production in the presence of μg ml - of quercetin decreased by more than % ± when compared to the non-treated infected cells (figure b ). daidzein showed very weak anti-dengue activity with ic = . μg ml - when the infected cells were treated after denv- adsorption (figure a ). its related si value was . . the levels of denv- rna production in the presence of μg ml - of daidzein decreased by only . % ± . when compared to the non-treated infected cells (figure b ). naringin and hesperetin did not exhibit any anti-dengue activity when they were used after adsorption of denv- to the vero cells ( figure ). although there was no significant direct virucidal activity against denv- by quercetin, continuous treatment of cells from h before virus infection up to days post-infection exhibited anti-dengue activity with ic = . μg ml - (figure a) . the si value for continuous treatment with quercetin was . and higher than the si value ( . ) for post-adsorption assay. in addition, the level denv- rna production decreased by more than . % ± . when vero cells were treated with μg ml - of quercetin, h before virus infection and up to days post infection ( figure b ). there was no significant change in the antiviral activity of daidzein when cells were treated continuously from h before virus infection up to days post infection comparing to its anti-dengue activity for postadsorption treatment (figure ). no significant antiviral activity for naringin and hesperetin was observed for the continuous treatment against denv- ( figure ). however, naringin exhibited anti-adsorption activity when it was added to the cells at the same time of virus adsorption. the ic value for naringin was . μg ml - and its related si was . ( figure a ). there was a reduction of . % ± . in denv- rna production in the presence of μg ml - of naringin (figure a) . the majority of the viral foci in cells treated with μg/ml quercetin appeared smaller, less intensely stained and more diffused within the focus (figure b) , compared to the larger, well-defined and more intensely stained foci of the untreated cells (figure a ). this observation is consistent with the reduction of the percentage of foci and rna copy number. results from the direct virucidal activity evaluation of each compound showed that there was no extracellular inhibitory activity against denv- for all the tested figure anti-adsorption activity of flavonoids against denv- . flavonoids were added directly to virus inocula for h at c. the inocula were used to infect vero cell monolayers in wells cell culture microplates. the reduction in foci forming unit was calculated relative to the controls maintained in parallel (a) and the respective denv- rna copy numbers were quantified using qrt-pcr (b). data from triplicate experiments were plotted using graph pad prism version (graph pad software inc., san diego, ca). the reduction of intracellular replication of dengue virus by . % and % following treatment with μm of glabranine and -o-methyle glabranine, respectively [ ] . similarly, other synthetic flavonoid derivatives also showed antiviral activity in hepg cells [ ] . whereas, pinostrobin was reported to inhibit denv- ns b/ns protease an enzyme important in dengue virus replication in an in vitro study [ ] . these suggest that flavonoids as a group could consist of select compounds that possesses inhibitory activities against denv. to investigate which of the many flavonoids could affect denv infection, in the present study, we examined the potential effects of quercetin, naringin, hesperetin and daidzein on dengue virus infection of vero cells. unlike the previous studies which evaluated antiviral activity of flavonoids only after adsorption of virus to the cells [ , ] , the present study evaluated antiviral activity by different treatment procedures tailored to determine prophylactic, post adsorption, continuous treatment and direct virucidal activities of quercetin, naringin, daidzein and hesperetin. our findings demonstrated that quercetin was the only compound among all tested flavonoids that consistently showed significant antiviral activity against denv- in vero cells. selectivity indices for quercetin when the infected cells were treated or when uninfected cells were treated continuously h before infection until days post-infection were . and . , respectively. the noted differences between si values for quercetin could be due to the intracellular accumulation of quercetin during continuous treatment. a weak effect for prophylactic activity of quercetin however, was also observed. these findings suggest that the main anti-dengue activity of quercetin is likely due to its activity against the different stages of intracellular replication of denv- instead of early stages of its replication cycle such as virus attachment or entry. although, no direct virucidal activity or anti-attachment activity of quercetin was observed in the present study, antiviral activity of quercetin against human cytomegalovirus was reported with ic = . μm [ ] . quercetin was also reported to be effective against herpes viruses where it is more specific against hsv- with si = compared to hsv- with si = . [ ] . the mechanisms of how quercetin exerts its antiviral effects are not known. however, the effects of other flavonoids against cellular rna polymerases and formation of the complex with rna have been reported suggesting that quercetin could also affect the similar replication enzymes [ , ] . sylimarin, a flavonoid found effective against hepatitis c virus (hcv), another member of flaviviridae family [ ] , inhibits virus replication by inhibiting the activity of viral rna polymerase. in our study, results from the qrt-pcr supported the findings from the viral foci reduction assays that quercetin inhibits denv- replication and the significant reduction in the denv specific rna suggests that quercetin may also target the virus replication machinery, namely by inhibiting the rna polymerase. antiviral activity of naringin has been evaluated against few herpesviruses and rotavirus but their reported antiviral activities against hsv- and hsv- are inconclusive [ , ] . in addition it was reported that naringin did not exhibit any antiviral activity against another rna virus, sindbis virus [ ] . in the present study, the only anti-dengue activity of this flavonoid was demonstrated against adsorption and attachment of virus to the vero cells and based on its antiviral activity (ic = . μg ml - ) and its related selectivity index (si = . ), it may not be a good candidate for further development as anti-dengue drug. similarly, daidzein activity against denv- was not significant compared to quercetin (si = . ). continuous treatment of the infected vero cells from h before virus infection up to days post infection did not improve its anti-dengue activity significantly. this compound therefore, could not be a suitable candidate for further development as anti-dengue drug. hesperetin, the other flavonoid evaluated in our study, did not show no anti-dengue activity in any stages of virus infection and replication processes and this is despite the previously reported antiviral activity of hesperetin against sindbis virus [ ] . therefore, hesperetin is also not recommended for further investigations for anti-dengue drug development. in all our experiments, we showed that . % of dmso, the highest concentration of solvent used in the bioflavonoid treatment did not exhibit any antiviral activity against denv- and this eliminated any probable antiviral activity from dmso. findings from our study, suggest that there are select flavonoids including quercetin and fisetin, which are both flavonol, that exhibited significant denv replication inhibition properties [ ] . while the flavonoids in general share common basic molecular base structure, flavone ( -phenyl- , -benzopyrone), we showed here that the flavanone, hesperetin, and flavanone glycoside, naringin, showed no significant anti-denv replication activities. in addition, we had earlier shown that naringenin [ ] , another flavanone metabolized from naringin and here, daidzein, an isoflavone also had no significant denv replication inhibition properties. while quercetin was shown here to be effective in inhibiting denv replication, its glycoside form, rutin (quercetin- -o-rutinoside) showed no significant inhibition properties [ ] . these suggest that while flavonol could be the basic molecule that possesses anti denv replication properties, specific structural properties of the different flavonol derivatives would have different effects on the efficacy of the compounds against dengue. the demonstration in vitro that flavonols including quercetin and fisetin possess anti denv replication properties does not necessarily translate into immediate use of these compounds as antivirals against denv. further studies will be needed to demonstrate the antiviral activities of these compounds against different genotypes of dengue virus and in appropriate animal model. there is also a need to address the issue of the low bioavailability of quercetin especially for therapeutic use [ ] [ ] [ ] . several strategies to increase the bioavailability of quercetin that include using lipids and emulsifiers, co-crystalization of quercetin or using ester-based precursors have been investigated [ ] [ ] [ ] . the other topic of research would be combination drug study. at its current calculated ic values, the antiviral efficacy of quercetin can be further improved possibly by combining it with other potential anti-dengue compounds. this is exemplified in a study that reported the synergistic effect of αglucoside in combination with a standard antiviral drug, ribavirin is effective against dengue infection [ ] . in conclusion, the present study demonstrates that the bioflavonoid quercetin exhibited significant anti denv replication properties. we further showed that quercetin affects intracellular denv virus replication but not the denv attachment and entry processes. these results together with the earlier findings reporting the anti denv properties of fisetin, suggest that these 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quercetin in rats cocrystals of quercetin with improved solubility and oral bioavailability ester-based precursors to increase the bioavailability of quercetin combination of α-glucosidase inhibitor and ribavirin for the treatment of dengue virus infection in vitro and in vivo antiviral activity of four types of bioflavonoid against dengue virus type- we thank the ministry of science, technology authors' contributions kz designed and carried out the antiviral and cytotoxicity studies and drafted the manuscript. btt carried out the virus propagation and antiviral studies. sss participated in the quantitative rt-pcr. wpf participated in the design of the study, performed statistical analyses and edited the manuscript. mrm participated in study design and provided all bioflavonoids. sab conceived the whole study and edited the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -db kb c authors: park, jun-gyu; Ávila-pérez, ginés; madere, ferralita; hilimire, thomas a.; nogales, aitor; almazán, fernando; martínez-sobrido, luis title: potent inhibition of zika virus replication by aurintricarboxylic acid date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: db kb c zika virus (zikv) is one of the recently emerging vector-borne viruses in humans and is responsible for severe congenital abnormalities such as microcephaly in the western hemisphere. currently, only a few vaccine candidates and therapeutic drugs are being developed for the treatment of zikv infections, and as of yet none are commercially available. the polyanionic aromatic compound aurintricarboxylic acid (ata) has been shown to have a broad-spectrum antimicrobial and antiviral activity. in this study, we evaluated ata as a potential antiviral drug against zikv replication. the antiviral activity of ata against zikv replication in vitro showed median inhibitory concentrations (ic( )) of . ± . μm and . ± . μm in vero and a cells, respectively; without showing any cytotoxic effect in both cell lines (median cytotoxic concentration (cc( )) > , μm). moreover, ata protected both cell types from zikv-induced cytopathic effect (cpe) and apoptosis in a time- and concentration-dependent manner. in addition, pre-treatment of vero cells with ata for up to h also resulted in effective suppression of zikv replication with similar ic( ). importantly, the inhibitory effect of ata on zikv infection was effective against strains of the african and asian/american lineages, indicating that this inhibitory effect was not strain dependent. overall, these results demonstrate that ata has potent inhibitory activity against zikv replication and may be considered as a potential anti-zikv therapy for future clinical evaluation. zika virus (zikv) belongs to the genus flavivirus within the flaviviridae family. zikv is an enveloped positive sense single-stranded rna virus with a genome size of ∼ . kb that encodes a single polyprotein, which is post-translationally processed by cellular and viral proteases into three structural (capsid, c; pre-membrane, prm; and envelope, e) and seven non-structural (ns , ns a, ns b, ns , ns a, ns b, and ns ) proteins avila-perez et al., ) . zika virus was initially isolated from uganda in and viral infections only occurred sporadically in africa and asia until . zikv appeared explosively as the first large-scale outbreak occurred in the yap island in and french polynesia in (weaver et al., ) . most recently, in , the first local transmission of zikv was found in territories of latin america and the caribbean, resulting in up to . million of zikv infection suspected cases (tang et al., ; tripathi et al., ) . like other members of the flaviviridae family, such as yellow fever virus (yfv), dengue virus (denv), japanese encephalitis virus (jev), and west nile virus (wnv), zikv is commonly transmitted by the bite of infected aedes mosquitos, but it can also be transmitted vertically from mother to child, through sexual contact, and in rare cases from blood transfusions (lessler et al., ; fink et al., ) . upon infection, zikv can be shed in blood, urine, semen, saliva, amniotic fluid, breast milk, and cerebrospinal fluid (nayak et al., ; colt et al., ; nazerai et al., ) . most people ( ∼ %) infected with zikv are asymptomatic or have mild symptoms such as fever, rash, joint pain, and conjunctivitis that can last for several days to a week (fink et al., ) . in rare cases, people with symptoms may have neurological guillain-barré syndrome complications (oehler et al., ; rivera-concepcion et al., ; nazerai et al., ) . in the case of pregnant women, zikv infection can lead to microcephaly and other fetal complications as occurred during the large-scale zikv outbreak in brazil in (lessler et al., ) . because of the significant outbreaks in south, central, and north america, zikv was declared a public health concern by the world health organization (who) in february (lazear and diamond, ; ramos da silva and gao, ; weaver et al., ; tripathi et al., ) . there are several vaccines and antiviral drugs currently under development for the prevention or treatment of zikv infection larocca et al., ; shan et al., ; fink et al., ) . dna-based larocca et al., ) , inactivated larocca et al., ; shan et al., ) , live-attenuated and mrna (richner et al., ) vaccines have been proposed for the prophylactic treatment of zikv infections. on the other hand, arbidol (arb) (fink et al., ; haviernik et al., ) , bortezomib, mycophenolic acid, daptomycin (barrows et al., ) , obatoclax, saliphenylhalamide, gemcitabine (kuivanen et al., ) , emetine (yang et al., ) , and sofosbuvir (bullard-feibelman et al., ) have been proposed for the therapeutic treatment of zikv infection. despite these tremendous efforts, there is currently no food and drug administration (fda)-approved vaccines and/or anti-viral drugs available for the treatment of zikv infection. since vaccination takes at least weeks to several months to show protective effects against zikv infection, vaccination is probably not the most appropriate prophylactic method for those who are traveling to areas where zikv is epidemic, endemic, or have already been infected. moreover, vaccination may cause an important issue, such as antibody-dependent enhancement (ade) priyamvada et al., ) . ade, which has been extensively described in denv (priyamvada et al., ) , is a phenomenon where preexisting antibodies facilitate binding and infection during subsequent exposure to infectious viruses, instead of neutralizing them, resulting in exacerbation of clinical signs priyamvada et al., ) . because of the structural similarities between denv and zikv, denv immunity-linked ade of zikv infection has also been reported priyamvada et al., ) . since vaccination for zikv could lead to denv ade, antivirals could represent a better choice for the control of zikv infection. aurintricarboxylic acid (ata), a polyanionic aromatic compound, has been shown to have inhibitory properties against several bacteria and viruses including, among others, yersinia pestis (liang et al., ) , cryptosporidium parvum (klein et al., ) , human immunodeficient virus (hiv) (mitra et al., ; de clercq, ) , hepatitis c virus (hcv) mukherjee et al., ; shadrick et al., ) , vaccinia virus (myskiw et al., ) , influenza virus (hung et al., ) , enterovirus (hung et al., ) and severe acute respiratory syndrome coronaviruses (sars-cov) (he et al., ) . mechanistic studies have suggested that ata has the ability to modulate various cellular enzymes such as activators of the janus kinase (jak ) and signal transducer and activator of transcription (stat ) families (rui et al., ) , inhibitors of nucleases (shadrick et al., ) , glucose- -phosphate dehydrogenase (bina-stein and tritton, ) , and topoisomerase ii proteins (catchpoole and stewart, ; benchokroun et al., ) as well as the enzymatic activity of the vaccinia virus ah l phosphatase (smee et al., ) . however, to date, the ability of ata to inhibit zikv infection has not been evaluated. herein, we investigated ata as a plausible prophylactic and therapeutic candidate against zikv infection. our results demonstrate that ata has a potent and effective antiviral activity against zikv in pre-and post-infection settings, including broadly antiviral activity against strains of the african and american/asian lineages with no toxicity up to , µm in cultured cells. these data support the feasibility of implementing ata for the treatment of zikv infection. african green monkey kidney epithelial vero (atcc ccl- ) and human adenocarcinoma alveolar basal epithelial a (atcc ccl- ) cells were maintained in dulbecco's modified eagle's medium (dmem; mediatech, inc.) supplemented with % fetal bovine serum (fbs) and % psg ( u/ml penicillin, µg/ml streptomycin, and mm l-glutamine) at • c in a % co atmosphere. paraiba/ zikv isolate was kindly provided by stephen dewhurst (department of microbiology and immunology, university of rochester). uganda/ (mr_ strain, catalog no. nr- ) and nigeria/ (ibh strain, catalog no. nr- ) zikv isolates were obtained from the biodefense and emerging infections research resources repository (bei resources). puerto rico/ (prvabc strain) and french polynesia/ zikv isolates were kindly provided from the centers for disease control and prevention (cdc). virus stocks were propagated in vero cells and titrated by plaque assay as previously described (marquez-jurado et al., ) . aurintricarboxylic acid (catalog no. a ) and arbidol (arb, catalog no. slm ) were purchased from sigma-aldrich, mo, united states. both compounds were prepared at mm stock solution dissolved in dimethyl sulfoxide (dmso) and kept at − • c until experimental use. each drug was diluted into infectious media (dmem % fbs, % psg) for the described experiments, where the maximum dmso concentration was . %. cell viability in vero and a cells was measured using the celltiter non-radioactive cell proliferation assay (promega) following the manufacturer's instructions. briefly, confluent vero or a cells ( -well plate format, × cells/well, triplicates) were treated with µl of dmem containing serially diluted (twofold dilutions, starting concentration of , µm) chemicals or . % dmso (vehicle control). plates were incubated at • c in a % co atmosphere for or h. samples were treated with µl of dye solution and incubated at • c in a % co atmosphere for h. next, cells were treated with µl of solubilization solution/stop mix and absorbance at nm was measured using a vmax kinetic microplate reader (molecular devices, waltham, ma, united states). viability of compound-treated cells was calculated as a percentage relative to values obtained with dmso-treated cells. non-linear regression curves and the median cytotoxic concentration (cc ) were calculated using graphpad prism software version . . confluent monolayers ( -plate format, × cells/well, triplicates) of vero cells were infected with plaque forming units (pfu)/well of paraiba/ , uganda/ , nigeria/ , puerto rico/ , and french polynesia/ at • c in infection media. after h of adsorption, virus inoculum was removed and cells were washed three times with infection media before adding fresh infection media containing % microcrystalline cellulose (avicel, sigma-aldrich) and the indicated concentration of compounds, or . % dmso as vehicle control. in case of pre-treatment experiments, the cell monolayers were treated with the indicated concentration of compound, or . % dmso, for the indicated times before zikv infection. infected cells were incubated at • c for - h, depending on virus strains. for immunostaining, cells were fixed with % paraformaldehyde for h, washed three times with phosphate buffered saline (pbs) and permeabilized with . % triton x- for min at room temperature. then, the plates were blocked with . % bovine serum albumin (bsa) in pbs (blocking solution) for h at room temperature, followed by incubation with µg/ml of the pan-flavivirus envelop (e) protein monoclonal antibody g (atcc, catalog no. vr- ) diluted in blocking solution for h at • c. after incubation with the primary antibody, cells were washed three times with pbs and developed with the vectastain abc kit and the dab peroxidase substrate kit (vector laboratory, inc., ca, united states) according to the manufacturers' instructions. stained plaques were analyzed using the ctl immunospot plate reader and counting software (cellular technology limited, cleveland, oh, united states). virus titers were calculated as pfu/ml (nogales et al., ) . non-linear regression curves and the median inhibitory concentration (ic ) were determined as described above. confluent monolayers ( -well plate format, . × cells/well, triplicates) of vero or a cells were infected (multiplicity of infection, moi, . ) with paraiba/ diluted in infection media for h at room temperature. after viral absorption, cells were incubated with infection media containing the indicated concentrations ( , , . , and µm) of ata. at , , , and h post-infection (h p.i.), tissue culture supernatants were collected and titrated on vero cells by immunostaining as described previously (marquez-jurado et al., ) . levels of apoptosis were measured using the caspase-glo r / assay (promega, wi, united states) following the manufacturer's instruction. briefly, vero and a cells ( -well plate format, . × cells/well, triplicates) were infected with zikv paraiba/ (moi of . ) and, at the indicated times post-infection, cells and tissue culture supernatants were collected and centrifuged. twenty five microliters of supernatants were mixed with µl of caspase- / reagent using a plate shaker, incubated at room temperature for h, and luminescence at nm was measured using a spectramax id (molecular devices, waltham, ma, united states) following the manufacturer's instructions. two-way anova was used to evaluate significant differences. data are expressed as the mean ± standard deviation (sd) of at least three independent experiments in triplicates using microsoft excel software. value were considered statistically significant when * p < . , * * p < . , * * * p < . , * * * * p < . . all data were analyzed with prism software version . (graphpad software, ca, united states). cc and ic were determined using sigmoidal dose response curves (graphpad software, ca, united states). the selective index (si) of each compound was calculated by dividing the cc with the ic . before examining the inhibitory effect of ata (figure ) against zikv infection, we first determined the cc of ata on vero and a cells (figure ) . for this, we treated both cell lines with serial (twofold) dilutions of ata and measured cell viability at and h post-treatment. as an internal control for these studies, we used arb, a drug that has been previously described to have antiviral activity against zikv in vero (haviernik et al., ) and a (fink et al., ) cells. we did not observe any toxicity with ata in vero (figure a ) or a ( figure b ) cells at or h post-treatment, even at the highest concentration tested ( , µm), while arb showed cc values of . ± . or . ± . µm in vero ( figure c ) and . ± . or . ± . µm in a ( figure d ) cells (table ) at or h post-treatment, respectively. to determine the ic of ata, vero and a cells were infected with pfu/well of paraiba/ and after h of viral absorption, virus inoculum was replaced with infection media with twofold serial dilutions (starting concentration of , µm) of ata or arb (figure ) and the ic calculated as described in the section "materials and methods." although the ic of ata ( figure a) and arb ( figure c) in vero cells were similar ( . ± . µm and . ± . µm, respectively), the selective index (si, cc /ic ) of ata (> . ) was significantly higher than that of arb ( . ) ( table ) . likewise, the ic of ata ( figure b ) and arb ( figure d ) in a cells were similar but with clearly different si values (> . for ata and . for arb) ( table ) . notably the cc , ic , and si of arb were similar to those previously described in the literature in these cell lines (fink et al., ; haviernik et al., ) . these data suggest that ata exhibited an effective inhibition of zikv infection with limited toxicity and si values better than those previously described for arb. we also observed that zikv replication was completely inhibited at a concentration of µm of ata in vero ( figure a ) and a ( figure b ) cells while . µm and µm concentrations of ata showed partial viral inhibition in vero and a cells (figures a,b) , respectively, demonstrating a dose-dependent inhibition of viral replication in both cell lines. we next evaluated the ability of ata to protect cells from the cytopathic effect (cpe) induced during zikv infection ( figure ) . to that end, vero and a cells were infected (moi . ) with paraiba/ and, after h of viral absorption, cells were treated with , . , , and µm of ata. at h p.i., cells were observed under a light microscope for evaluation of their morphology and cpe ( figure a ). as expected from our previous results, µm and more clearly µm of ata were able to prevent zikv-induced cpe in both cell lines (figure a) . to quantify the ability of ata to prevent zikv-induced apoptosis, tissue culture supernatants from zikv-infected vero and a cells were harvested at , , and h p.i. to measure the level of apoptotic signal as determined by caspase and activities ( figure b) . zikv-infected cells showed figure | aurintricarboxylic acid inhibition of zikv replication: vero (a) and a (b) cells ( -well plate format, . × cells/well, triplicates) were infected (moi . ) with paraiba/ . tissue culture supernatants were collected at , , and h p.i., and viral titer were calculated by immunostaining (fluorescent forming units, ffu/ml). dotted line indicates the limit of detection ( ffu/ml). data was expressed as mean and standard deviations (sd) from three independent experiments conducted in triplicates. statistical analysis was conducted by two-way anova, * p < . , * * p < . , * * * p < . , * * * * p < . , or no significance (n.s.). figure | aurintricarboxylic acid protects vero and a cells from zikv-induced cell death: vero and a cells ( -well plate format, . × cells/well, triplicates) were infected (moi . ) with paraiba/ . after h viral adsorption, cells were treated with the indicated concentrations ( , , . , and µm) of ata. at h p.i., cells were observed and imaged under an optical microscope. scale bar = µm. (a) caspase / levels were measured in the tissue culture supernatants at , , and h p.i. (b) data of each time point was compared to mock-infected control cells and expressed as mean of relative percentage and sd from three independent experiments conducted in triplicates. statistical analyses were conducted by two-way anova, * p < . , * * p < . , * * * p < . , * * * * p < . , or no significance (n.s.). increased caspase and levels up to eightfolds in vero cells and up to . -folds in a cells compared to mock-infected cells ( figure b ). levels of caspase and activation were dose-dependently reduced by ata with µm of ata showed only . -and . -fold induction as compared to mock-infected vero and a cells, respectively ( figure b ). we next determined whether ata is able to inhibit both ancestor african (uganda/ and nigeria/ ) and contemporary asian/american (puerto rico/ and french polynesia/ ) zikv lineage strains using our microplaque reduction assay (figure ) . we observed similar ic values of ata with uganda/ ( figure a , ic = . ± . µm), nigeria/ ( figure b , ic = . ± . µm), puerto rico/ ( figure c , ic = . ± . µm), and french polynesia/ ( figure d , ic = . ± . µm), compared to those observed with paraiba/ (figure and table ), demonstrating the broad antiviral activity of ata against different zikv strains, regardless of the year and place of isolation. to demonstrate the feasibility of using ata for the prevention of zikv infection, important for travelers to regions where zikv is endemic, we next evaluated whether pre-treatment with ata results in inhibition of zikv replication (figure ) . to that end, vero cells were pre-treated with ata for ( figure a) , ( figure b) , (figure c) , or ( figure d ) h prior to infection (moi . ) with paraiba/ . pre-treatment with ata for - h before zikv infection resulted in similar ic values ( . ± . µm, . ± . µm, . ± . µm, and . ± . µm; respectively) demonstrating that ata is stable and able to prevent zikv infection even when administered days previous to viral infection (figure and table ). the recent outbreak of zikv accompanied with severe pathology, including microcephaly in newborns, prompted many researchers to develop prophylactic vaccines and to identify therapeutic drugs against zikv infection larocca et al., ; lessler et al., ; shan et al., ; fink et al., ) . currently, there are no commercially available vaccines and/or antiviral therapies for the treatment of zikv infection. therefore, there is an urgent medical need for the development of effective counter measurements to control zikv infection. in this study, we demonstrated that ata (figure ) has limited toxicity (figure ) and an effective and dose-dependent antiviral activity against zikv infection (figures , ) in both monkey kidney epithelial vero and human alveolar a cells. notably, ata can prevent zikv-induced cpe and apoptosis in both cell lines ( figure ) and has broad anti-viral activity against representative zikv strains from the african (uganda/ and nigeria/ ) and the asian/american (puerto rico/ and french polynesia/ ) lineages (figure ) . moreover, ata can also prevent zikv infection even when administered days before infection (figure ) . aurintricarboxylic acid is a polyanionic aromatic compound that structurally relates to suramin (balzarini et al., ) and is believed to influence over host and viral enzymes (shadrick et al., ) . although the exact mechanism by which ata inhibits zikv infection was not identified in this study, there are several plausible mechanisms on zikv inhibition mediated by ata, including the targeting of viral and cellular proteins. in terms of inhibiting viral proteins, ata could bind to zikv ns helicase and prevent its binding to either atp or nucleic acids, as previously described for hcv (mukherjee et al., ; shadrick et al., ) . likewise, ata could inhibit the zikv rna-dependent rna polymerase (rdrp) ns protein, as described for hcv mukherjee et al., ; shadrick et al., ) and enterovirus (hung et al., ) . similarly, ata could inhibit the methyltransferase activity of ns involved in mrna capping processes, as previously described for other flaviviruses (denv and yfv) (milani et al., ; garcia et al., ) . because of the structural similarities between denv and zikv ns proteins, it is feasible that, similar to denv, ata binds to ns to inhibit zikv infection . moreover, it is possible that ata targets and has inhibitory activities against one or more of the viral proteins described above. in terms of targeting cellular proteins important for the efficient replication of zikv, it has been previously described that ata has anti-apoptotic properties in a variety of cells (chen et al., ) . it is possible that the anti-apoptotic activity of ata protects against zikv-induced cell death, as demonstrated in this study (figure ) . notably, it has been recently shown that zikv infection induced apoptosis through caspase and in a cells and through caspase in neonatal mice brain (huang et al., ; frumence et al., ) . these results suggest that inhibition of zikv replication results in a decrease in the level of apoptotic cells and that the anti-apoptotic effect of ata affects zikv replication. further research is guaranteed to yield a better understanding of the antiviral activity of ata on zikv infection, and other viruses, before the use of ata as an antiviral drug. during january to february , a total of residents from states in the united states were diagnosed with zikv infection (armstrong et al., ) . out of patients, ( %) traveled to areas of active zikv transmission before the infection and five ( %) did not travel but reported sexual contact with a traveler who had a symptomatic illness (armstrong et al., ) . for these reasons, preventive efforts are required prior to travel to areas of active zikv transmission. in this study, cells pretreated with ata for up to h prior to infection with zikv showed similar ic than those in post-treatment settings, potentially suggesting that ata might target a cellular protein required for zikv replication or that the concentration and stability of ata in pre-treated cells is sufficient to inhibit zikv infection, or both. nevertheless, these results demonstrate the feasibility of using ata for the prophylactic treatment of viral infection, including those traveling to areas where zikv is endemic. moreover, due the broad inhibition effect of ata against others viruses and parasites (liang et al., ; he et al., ; de clercq, ; myskiw et al., ; klein et al., ; chen et al., ; hung et al., hung et al., , mukherjee et al., ; shadrick et al., ) that are present in zikv endemic areas, treatment with ata could be used for the broad prevention of denv, yfv (milani et al., ; shadrick et al., ; garcia et al., ) , hcv mukherjee et al., ; shadrick et al., ) , and parasitic infestation (cryptosporidium parvum) (klein et al., ) for people traveling to these endemic regions. moreover, the broad spectrum antiviral activity of ata against different african and asian/american zikv strains further guarantees the feasibility of implementing ata to prevent zikv infection to travelers around the world. although ata has been amply evaluated in vitro, only few studies have assessed the activity of ata in vivo, including its use as a curative agent against thrombosis (strony et al., ) , apoptosis (roberts-lewis et al., ; heiduschka and thanos, ) , parasite infestations (klein et al., ) , bacterial (y. pestis) (liang et al., ) , and vaccinia virus (smee et al., ) infections. in the case of vaccinia virus, ata did not protect mice from a lethal challenge at a dose of mg/kg/day (smee et al., ) . further studies are needed to evaluate the anti-viral activity of ata in vivo for the treatment of viral infections, including zikv. our studies show limited toxicity, if any, of ata in cultured cells, including human a cells. the lack of knowledge about the use of ata in pregnant women requires future additional safety tests, including studies using validated animal models of zikv infection, before using ata for the treatment of zikv infection during pregnancy. based on the effectiveness of ata against zikv infection (si = . in vero cells and . in a cells) as compared to other previously described drugs, including emetine (si = . in snb- cells and . in env+ cells) (yang et al., ) , obatoclax [si = in human retinal pigment epithelial (rpe) cells] (kuivanen et al., ) , saliphenylhalamide (si > in rpe cells) (kuivanen et al., ) , gemcitabine (si > , in rpe cells) (kuivanen et al., ) , sofosbuvir (si > . in huh- cells and . in jar cells) (bullard-feibelman et al., ) and arb (si = . in vero cells and . in a cells) (fink et al., ; haviernik et al., ) and this study (ata, in vero cells, and . in a cells), it is possible that ata represents one of the most reasonable options of the treatment of zikv infection. protective efficacy of multiple vaccine platforms against zika virus challenge in rhesus monkeys travel-associated zika virus disease cases among u.s. residents-united states reverse genetic approaches for the generation of recombinant zika virus aurintricarboxylic acid and evans blue represent two different classes of anionic compounds which selectively inhibit the cytopathogenicity of human t-cell lymphotropic virus type iii/lymphadenopathy-associated virus enhancement of zika virus pathogenesis by preexisting antiflavivirus immunity a screen of fda-approved drugs for inhibitors of zika virus infection aurintricarboxylic acid, a putative inhibitor of apoptosis, is a potent inhibitor of dna topoisomerase ii in vitro and in chinese hamster fibrosarcoma cells aurintricarboxylic acid is a nonspecific enzyme inhibitor the fda-approved drug sofosbuvir inhibits zika virus infection inhibition of topoisomerase ii by aurintricarboxylic acid: implications for mechanisms of apoptosis inhibition of cytokine-induced jak-stat signalling pathways by an endonuclease inhibitor aurintricarboxylic acid characterization of aurintricarboxylic acid as a potent hepatitis c virus replicase inhibitor transmission of zika virus through breast milk and other breastfeeding-related bodily-fluids: a systematic review emerging anti-hiv drugs the antiviral drug arbidol inhibits zika virus the south pacific epidemic strain of zika virus replicates efficiently in human epithelial a cells leading to ifn-beta production and apoptosis induction inhibitors compounds of the flavivirus replication process arbidol (umifenovir): a broad-spectrum antiviral drug that inhibits medically important arthropod-borne flaviviruses potent and selective inhibition of sars coronavirus replication by aurintricarboxylic acid aurintricarboxylic acid promotes survival and regeneration of axotomised retinal ganglion cells in vivo zika virus infection during the period of maximal brain growth causes microcephaly and corticospinal neuron apoptosis in wild type mice inhibition of enterovirus replication and the viral d polymerase by aurintricarboxylic acid aurintricarboxylic acid inhibits influenza virus neuraminidase in vitro and in vivo activity of aurintricarboxylic acid preparations against cryptosporidium parvum obatoclax, saliphenylhalamide and gemcitabine inhibit zika virus infection in vitro and differentially affect cellular signaling, transcription and metabolism vaccine protection against zika virus from brazil zika virus: new clinical syndromes and its emergence in the western hemisphere assessing the global threat from zika virus aurintricarboxylic acid blocks in vitro and in vivo activity of yoph, an essential virulent factor of yersinia pestis, the agent of plague an alanine-to-valine substitution in the residue of zika virus ns a protein affects viral rna synthesis and attenuates the virus in vivo flaviviral methyltransferase/rna interaction: structural basis for enzyme inhibition hiv- upregulates fas ligand expression in cd + t cells in vitro and in vivo: association with fas-mediated apoptosis and modulation by aurintricarboxylic acid identification and analysis of hepatitis c virus ns helicase inhibitors using nucleic acid binding assays aurintricarboxylic acid inhibits the early stage of vaccinia virus replication by targeting both cellular and viral factors pathogenesis and molecular mechanisms of zika virus a 'furrytale' of zika virus infection: what have we learned from animal models? replication-competent influenza a viruses expressing a red fluorescent protein zika virus infection complicated by guillain-barre syndrome-case report, french polynesia humoral immune responses against zika virus infection and the importance of preexisting flavivirus immunity zika virus: an update on epidemiology, pathology, molecular biology, and animal model modified mrna vaccines protect against zika virus infection the zika virus: an association to guillain-barre syndrome in the united states -a case report aurintricarboxylic acid protects hippocampal neurons from nmda-and ischemia-induced toxicity in vivo activation of the jak -stat signaling pathway in nb lymphoma cells by an anti-apoptotic agent, aurintricarboxylic acid aurintricarboxylic acid modulates the affinity of hepatitis c virus ns helicase for both nucleic acid and atp a live-attenuated zika virus vaccine candidate induces sterilizing immunity in mouse models lack of efficacy of aurintricarboxylic acid and ethacrynic acid against vaccinia virus respiratory infections in mice aurintricarboxylic acid in a canine model of coronary artery thrombosis zika virus infects human cortical neural progenitors and attenuates their growth a novel zika virus mouse model reveals strain specific differences in virus pathogenesis and host inflammatory immune responses zika virus: history, emergence, biology, and prospects for control emetine inhibits zika and ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry key: cord- -tibtngy authors: muñoz-moreno, raquel; cuesta-geijo, miguel Ángel; martínez-romero, carles; barrado-gil, lucía; galindo, inmaculada; garcía-sastre, adolfo; alonso, covadonga title: antiviral role of ifitm proteins in african swine fever virus infection date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: tibtngy the interferon-induced transmembrane (ifitm) protein family is a group of antiviral restriction factors that impair flexibility and inhibit membrane fusion at the plasma or the endosomal membrane, restricting viral progression at entry. while ifitms are widely known to inhibit several single-stranded rna viruses, there are limited reports available regarding their effect in double-stranded dna viruses. in this work, we have analyzed a possible antiviral function of ifitms against a double stranded dna virus, the african swine fever virus (asfv). infection with cell-adapted asfv isolate ba v is ifn sensitive and it induces ifitms expression. interestingly, high levels of ifitms caused a collapse of the endosomal pathway to the perinuclear area. given that asfv entry is strongly dependent on endocytosis, we investigated whether ifitm expression could impair viral infection. expression of ifitm , and reduced virus infectivity in vero cells, with ifitm and ifitm having an impact on viral entry/uncoating. the role of ifitm in the inhibition of asfv in vero cells could be related to impaired endocytosis-mediated viral entry and alterations in the cholesterol efflux, suggesting that ifitm is acting at the late endosome, preventing the decapsidation stage of asfv. upon infection with pathogens such as bacteria or viruses, the host cell activates the innate immune response as a first line of defense. the group of cytokines known as interferons (ifn) plays a major role in the cell immunity by inducing a cascade of interferon-stimulated genes (isgs) that encode for several antiviral innate immune effectors. among isgs, the interferoninduced transmembrane proteins (ifitms) are known to inhibit entry of a wide variety of enveloped rna viruses [ ] . this group of proteins is present across a wide range of species: from amphibians, fish and birds to mammals. ifitms in humans were identified years ago as interferon-stimulated genes upon induction of type-i and type-ii ifn [ , ] . human ifitm , ifitm and ifitm are expressed in almost every cellular type, whereas ifitm is expressed primarily in osteoblasts, as it is required for bone mineralization [ ] . ifitms are found mainly distributed at the plasma membrane and/or at endosomal membranes. the ifitm , , and genes are clustered on chromosome , and they encode for relatively small proteins (about amino acids) with both extra-cytoplasmic termini separated by two transmembrane domains (tm and tm ) and a cytoplasmic loop (cil) [ ] [ ] . tm and the cil are well conserved between the ifitm proteins and a large group of members of the cd protein family. ifitm , , and are currently known to inhibit the replication of multiple rna viruses that enter the host cell via endocytosis, including influenza a virus (iav), west nile virus (wnv), dengue virus (denv) [ ] , severe acute respiratory syndrome coronavirus (sars cov) and hepatitis c virus (hcv) [ ] . in contrast, ifitms do not inhibit the entry process of mouse leukemia virus (mlv), machupo virus (mach), lassa virus (lasv) or lymphocytic choriomeningitis virus (lcmv) [ ] . little is known about the ifitm-mediated antiviral activity against dna viruses. only ifitm has been recently described to inhibit rana grylio virus (rgv), a frog/fish iridovirus, at the entry stage [ ] . on the other hand, ifitm , and have been reported not to affect the replication of other dna viruses, such as human papillomavirus (hpv), human cytomegalovirus (hcmv) and adenovirus (ad ) [ ] . the antiviral effect of ifitms is mainly exerted through their effects on the endocytic pathway and would affect viruses entering the cell through a late endosomal compartment [ ] . to further expand our understanding on the antiviral activity of ifitms against dna viruses, we investigated the role of these proteins in the replication cycle of the african swine fever virus (asfv), belonging to the nucleocytoplasmic large dna virus (ncldv) superfamily [ ] . asfv infection is strongly dependent on the endocytic pathway [ , ] , thus a possible ifitm-mediated inhibition of the virus could likely occur in the endosomal compartments. asfv is the only member of the asfarviridae family and is responsible of a highly lethal and hemorrhagic disease affecting domestic swine, which often results in important economic losses in many countries due to the high rate of mortality associated with the illness and the lack of an effective vaccine [ ] . an epidemic outbreak is currently affecting east europe and is slowly spreading between neighboring countries [ ] [ ] [ ] . we previously reported that asfv enters into the host cell by dynamin-dependent and clathrin-mediated endocytosis [ , ] . thus, our goal in the current work was to test whether the ifitm family of proteins affected early entry steps of asfv infection in vero cell cultures using the cell-adapted ba v isolate. vero cells were obtained from the american type culture collection (atcc, richmond, va, usa) and maintained in dulbecco modified eagle medium supplemented with % fetal bovine serum (fbs), lu/ml penicillin, ug/ml streptomycin and mm l-glutamine at °c at % co . cells were pretreated with , or , u/ml of universal type-i ifn (pbl assay science) for h, as indicated. the tissue culture-adapted asfv isolate ba v was used in most experiments [ ] . for flow cytometry analyses, a recombinant virus expressing the viral protein p fused to the green fluorescent protein (gfp) was used (b gfp) [ ] . preparation of viral stocks, titrations and infection procedures were carried out in vero cells as previously described [ ] . when synchronization of viral infection was required, the adsorption phase took place at °c to allow viral attachment to the cell surface but impeding its internalization. when indicated, asfv was semi-purified by sucrose cushion ( %) in pbs at , xg for min at °c. to generate stable cell lines expressing different proteins, the commercially available lentiviral expression vector plvx-puro (clontech) was used to clone the proteins of interest. t cells were transfected at % confluency using lipofectamine (life technologies) with opti-mem (life technologies) in -cm plates. plates were previously pretreated with poly-l-lysine (sigma-aldrich) at a final concentration of . mg/ml to avoid cell detachment. co-transfection of plvx-puro expression vector together with vesicular stomatitis virus glycoprotein (vsv-g) and the human immunodeficiency virus (hiv) gag-pol expressing plasmids was performed to produce pseudotyped lentiviral vectors. supernatants containing the pseudotyped lentiviruses were collected twice at h and h postransfection. cell debris was removed by brief centrifugation at , rpm for min and cleared supernatants were . μm-filtered and stored at - °c until use. sub-confluent vero cells were transduced with the pseudotyped lentiviruses expressing the gene of interest and supplemented with μg/ml of polybrene (emd millipore). h later, transduced cells were selected by adding μg/ml of puromycin (life technologies). optimal puromycin working concentration was previously titrated in non-transduced cells. finally, protein expression levels of vero stable-cell lines were determined by western blot (wb). cells were seeded and grown on glass coverslips. mock-infected and infected cells were fixed with % paraformaldehyde in pbs for min at room temperature (rt). following cell fixation, aldehyde fluorescence was quenched by incubation of cells with mm nh cl in pbs for min. then, cells were permeabilized with pbs- . % triton x- or saponin (sigma) for min at rt. after blocking with bovine serum albumin (bsa; sigma) or normal goat serum (sigma), cells were stained with primary and secondary antibodies and then incubated with topro- (molecular probes) in pbs at a : , ratio for dna staining. after washing, coverslips were finally mounted on glass plates and cells were observed under a leica tcs sp -aobs confocal microscope (leica-microsystems, wetzlar, germany) using a x immersion oil objective. to detect cholesterol distribution we used filipin staining (sigma), as previously described [ ] . cholesterol was visualized in a conventional leica dm rb microscope by combining a x immersion oil objective and a uv filter set. images were captured with leica application suite advanced fluorescence software (las af) and imagej software. finally, digital images were processed with adobe photoshop . . the primary antibodies used for immunofluorescence assays included the following: rabbit polyclonal antibodies to ifitm , ifitm and ifitm (proteintech), : ; anti-asfv p mouse monoclonal antibody, : (kindly given by dr. j.m. escribano, inia); asfv mouse monoclonal antibodies anti-p (clone bc for immunofluorescence : , or clone bg for wb : , ) and anti-p (clone ah , ingenasa), : , ; mouse monoclonal to cd (developmental studies hybridoma bank, university of iowa, clone h c ), : ; rabbit polyclonal to lamp (abcam), : ; rabbit polyclonal to eea and rab (cell signalling), : . secondary antibodies were purchased from molecular probes and diluted : . specificity of labeling and absence of signal crossover were determined by examination of single labeled control samples. cells were harvested in sample buffer laemmli x concentrate (sigma). then, the samples were incubated for min at °c and resolved by sds-page in % or % polyacrylamidebisacrylamide gels. afterwards, separated proteins were transferred to a nitrocellulose membrane (bio-rad) and the non-specific antibody binding sites were blocked with skimmed milk diluted in pbs and then incubated with the specific primary and hrp (horseradish peroxidase)-conjugated secondary antibodies. antibodies used for western blotting included: rabbit polyclonal to ifitm , ifitm and ifitm (proteintech), : , ; anti-asfv p mouse monoclonal antibody, : ; anti-asfv p mouse monoclonal antibody (clone bc , ingenasa), : , , and mouse monoclonal to tubulin (sigma-aldrich), : , . as secondary antibody, anti-mouse igg (ge healthcare) or anti-rabbit igg (bio-rad) conjugated to horseradish peroxidase was used at a : , dilution. precision protein streptactin-hrp conjugate (bio-rad) was used to reveal the ladder precision plus protein westernc (bio-rad). as a loading control an anti-mouse antibody against β-tubulin (sigma) was used, : , . finally, bands obtained after development with ecl reagent (ge healthcare, piscataway, nj, usa) were detected using a chemidoc xrsplus imaging system (bio-rad). band densitometry was performed with image lab software (bio-rad) and normalized to control values. the quantitation of the number of copies of asfv genome was achieved by quantitative realtime pcr (qpcr) using specific oligonucleotides and a premix extaq (tm) ( x; takara) probe. fluorescent hybridization probes were used to amplify a region of the p viral gene, as described previously [ ] . dna from cells mock-infected or infected with asfv ba v at moi of pfu/cell was extracted at hpi and purified with a dnaeasy blood and tissue kit (qiagen). dna concentration was measured using nanodrop. the amplification mixture was prepared on ice as follows: ng template dna diluted in mq h o to a total volume of μl, μl oligonucleotide oe f ( pmol), μl oligonucleotide oe r ( pmol), μl pcr premix ex taq(tm) ( x; takara), μl taqman probe se ( pmol) [ ] . positive amplification controls included dna purified from asfv purified virions at different concentrations as standards. negative amplification controls consisted in dna from mock-infected cells. each sample was included in triplicates and values were normalized to standard positive controls. reactions were performed using the abi fast real-time pcr system (applied biosystems) with the following parameters: cycle at °c for min, cycles at °c for s, and cycles at °c for min. to study virion decapsidation in asfv, a protocol was adapted from a previous publication [ ] . briefly, stable cell lines vero-ifitm , ifitm , ifitm or control cells containing the empty vector were infected at moi of pfu/cell after viral synchronization at °c for minutes to enable virus attachment to the cell but restricting viral entry. infection was allowed to proceed for minutes at °c, % co . cells were then washed with cold pbs x and treated with . % trypsin-edta (gibco) for minutes at °c to remove the membrane-bound virus. finally, cells were placed in media containing fcs to quench trypsin activity and washed with pbs. after this treatment, only internalized virions were observed in an immunofluorescence assay as described above. we used specific antibodies to detect the major viral capsid protein p and the viral core protein p , and staining was analyzed by confocal microscopy. decapsidated virions were single labeled for p and were counted for each condition and normalized to the total number of fully encapsidated virions which were double labeled for p and p . stable cell lines vero-ifitm , ifitm , ifitm or control cells containing the empty vector were infected with ba v or b gfp at the indicated moi. recombinant b gfp is a recombinant asfv expressing green fluorescent protein as a fusion protein of viral p [ ] . samples infected with b gfp at hpi were just fixed and washed with facs buffer three times before analysis. vero cells infected with ba v at hpi or hpi (early or late postinfection times respectively), were harvested by trypsinization, washed with facs buffer (containing pbs, , % sodium azide, and , % bovine serum albumin), fixed and permeabilized with perm (bd science) for min at rt and finally incubated with specific primary antibodies against p and p for min at °c. the secondary antibody used was phycoerythrin (pe) conjugated (dako) : diluted in facs buffer for min at °c. after repeated washes, , cells/ tube were analyzed in the facscalibur flow cytometer (bd science) in triplicates. the obtained infection rates were always normalized to the corresponding control. bonferroni's multiple-comparison test was used to compare different experimental groups. prism software (graphpad software, inc.) and instat software were used for the statistical analysis. values were expressed in graph bars as mean ±sd of at least three independent experiments unless otherwise noted. metrics were normalized to control values and represented in graphics. asterisks denote statistically significant differences ( ÃÃà p< . , Ãà p< . and à p< . ). to determine the effect of interferon on asfv infection, vero cells were pretreated with , or , u/ml of universal type-i ifn (pbl assay science) and h later, they were infected with recombinant asfv b gfp at a moi of pfu/cell (fig ) . viral infection was quantified by analyzing the number of gfp-positive cells by flow cytometry at hpi (fig a) . pretreatment of vero cells with universal type-i ifn at both concentrations completely abrogated asfv infectivity when compared to untreated control cells. a sample flow cytometry profile is shown (fig a) . ifn treatment induces expression of ifitm proteins ifitm proteins are located in different cellular compartments and their antiviral properties strongly correlate with their capacity to alter the fluidity and fusion ability of the membranes in which they reside [ , ] . then, we analyzed ifitms expression and distribution in vero cells upon ifn treatment. to this end, cells were incubated with either , or , u/ml of universal type-i ifn for h and ifitms , and distribution was analyzed by confocal microscopy ( fig b) . although basal levels of ifitm and were detected prior to treatment with to further analyze the expression of ifitm , and upon ifn treatment, vero and t cells were incubated with universal type-i ifn for h and analyzed by wb (fig ) . while ifitm and ifitm were induced by ifn in both cell lines (fig a and b) , ifitm showed the highest induction (fig c) . in order to analyze the possible impact of ifitms in asfv infection, we generated vero cells stably expressing the human ifitm , , (hereinafter referred to as vero-ifitm , or cells respectively) or control cells containing the empty vector. to generate the stable cell lines we used a lentiviral transduction system. our proteins of interest were cloned into the plvx vector (see material and methods section for detailed experimental procedures). positively transduced vero stable cells were selected with μg/ml of puromycin. once these cells were established, the expression of different ifitm proteins was confirmed by wb analysis ( fig a) . as shown in the corresponding wb densitometry (fig b) , the highest expression levels within the ifitm family members corresponded to ifitm , followed by ifitm and ifitm . after assessing the expression levels of the vero-ifitm cells, we next wanted to ascertain the subcellular distribution of each ifitm. expression of ifitm , and in vero-ifitm cells was compared with the distribution of ifitms in vero cells with the empty vector. confocal microscopy experiments revealed that, ifitm was mainly distributed at the plasma membrane and to a lesser extent in perinuclear compartments, resembling endosomal structures (fig c, lower left panel) , while endogenous ifitm was barely detected in vero cells containing the empty vector (fig c, upper left panel) . in vero-ifitm cells, overexpression led to a high accumulation of the protein in the perinuclear region, colocalizing with vesicular structures that resembled endosomes (fig c, medium lower panel). consistent with fig b, there was a significant expression of endogenous ifitm in control vero cells containing the empty vector, which displayed a mitochondrialike pattern together with vesicular-like structures. finally, overexpressed ifitm was found predominantly accumulated in the perinuclear region, showing a pattern consistent with localization at clustered endosomal structures ( fig c, lower right panel) . endogenous ifitm was barely detected in vero cells containing the empty vector (fig c, upper right panel) . analysis of endogenous ifitm expression in control vero cells suggested a mixed mitochondrial and vesicular pattern. to confirm this observation, we analyzed subcellular localization of ifitm using mitotracker red as a specific marker for mitochondria. confocal images revealed a distribution of ifitm to mitochondrial structures in cells containing the empty vector (fig ) . however, in vero-ifitm cells, ifitm labelling localized to endosomal-like structures and we found few areas of colocalization between ifitm and mitochondria (fig b) . then, we investigated the possible mechanism underlying the inhibition caused by ifitm in the viral infection. to further analyze the vesicular localization of ifitms, we studied the cell. data are expressed as mean±sd of three independent experiments. statistical significance was evaluated by one-way anova followed by bonferroni's multiple comparison test. differences are marked with asterisks as indicated (***p< . ). an example of a typical facs profile is shown. (b). confocal fluorescence images of ifitm , and subcellular distribution in untreated vero cells or upon increasing universal ifn concentrations ( , or , u/ml) for h. bar = μm. (fig a) . endosomes are normally distributed through the cytoplasm and this dispersed distribution was found for the different maturation stages of endosomes in controls and in vero-ifitm cells. however, in vero-ifitm and ifitm cells dispersed distribution changed and endosomes aggregated around the nucleus (fig a) . this endosome redistribution was analyzed by confocal microcopy in x, y, z planes by measuring the mean distance between each endosomal marker and the cell nucleus, using the "distance measure" imagej plug-in (fig b) . a total of cells were analyzed for each condition. we concluded that overexpression of ifitm and ifitm altered endosome distribution by accumulating these vesicles to the perinuclear region, similar to the pattern previously found after ifn treatment of vero cells (fig b) and this redistribution might reflect alterations in endo-lysosomal maturation and function. next, we analyzed the colocalization rate between ifitms and endosomal structures. cd remains mainly associated with intracellular vesicular membranes and it is particularly abundant in endosomal structures called multivesicular bodies (mvbs), which constitute a late and acidic endosomal compartment filled with intraluminal vesicles (ilvs). these ilvs are filled with cholesterol and are crucial for endosomal membrane backfusion and necessary for late endosome maturation. co-staining of ifitms and cd by immunofluorescence assay revealed a clear colocalization of ifitms and cd in vero-ifitm cells, with % colocalization for vero-ifitm cells and % in vero-ifitm and in ifitm cells (fig a) . then, ifitm , and to a lesser extent ifitm and , were located primarily in endosomal compartments as indicated by higher colocalization with endosomal marker cd (fig c) when compared to the empty vector ( fig b) . ifitm proteins could reduce curvature of cell membranes for fusion pore formation [ , ] at the outer endosomal membrane, also called endosomal-limiting membrane, to differentiate it from the membranes of intraluminal vesicles inside multivesicular bodies (mvb/le). this would lead to an alteration of membrane fusion and impaired cholesterol endosomal efflux. changes in endosomal distribution such as those found in ifitm stably expressing cells (fig ) is a characteristic phenotype of an altered cholesterol endosomal efflux [ , ] . conversely, a conserved endosomal cholesterol efflux is required for a correct endosomal function. therefore, we analyzed intracellular and intra-endosomal cholesterol levels by using the cholesterol marker filipin. vero-ifitm and ifitm cells showed intense intracellular cholesterol accumulation at the perinuclear area ( fig a) that was absent in control cells containing the empty vector (fig b) . this cholesterol accumulation also colocalized with the ifitm-labeled endosomal vesicles. in contrast, in vero-ifitm cells, only discrete colocalization areas between ifitm and cholesterol were found at the plasma membrane. collectively, these findings indicate that ifitm and ifitm overexpression in vero cells results in cholesterol accumulation in endosomal compartments, and as a result it might be responsible of an altered endosomal function possibly altering viral entry through this pathway. next, we investigated whether overexpression of ifitms could restrict asfv infection or not. asfv is known to require acidic endosomal compartments for entry into host cells. successful asfv entry and egress from endosomes depends on the acidic ph of late endosomes [ ] . previous experiments in the laboratory revealed that the inhibition of acidification impaired viral decapsidation and viral particles were retained in rab -positive late endosomes, thus blocking viral infection progression [ ] . given that ifitm restriction could be mediated at the endocytic pathway [ , ] , we hypothesized that ifitm overexpression may affect virus entry process and subsequent asfv infection. acidic ph of late endosomal compartments is required for viral decapsidation, which is the first step required for uncoating previous to endosomal escape of the virus and the start of replication [ ] . the asf virion is composed of several concentric domains. the viral capsid is formed by major capsid protein p organized in capsomers. hence, after decapsidation, p staining of virions is lost. the internal core is composed by the nucleoid containing genome coated by the core shell, a thick protein layer that can be labeled with antibodies against p , a core shell protein derived from processed core shell polypeptides [ ] . . (b) . the change in distribution was quantified by measuring the mean distance to the nucleus of the different endosomal markers in x, y and z planes as described in materials and methods section. as shown in graphics, distance was reduced in vero-ifitm and cells. graphics depict mean±sd of n = cells per condition. statistical significance was evaluated by a one-way anova followed by bonferroni's multiple comparison test. differences are marked with asterisks as indicated (*p< . ; **p< . ). doi: . /journal.pone. .g we used antibodies against the major viral capsid protein p to detect viral capsids and against viral core protein p to detect viral cores (fig ) . we analyzed the number of encapsidated viral particles, double labeled for p and p , and compared with the number of successfully decapsidated viral cores positive for p at minutes postinfection (mpi). at this time point, most virions undergo uncoating and progress to replication in normal conditions, otherwise, encapsidated virions would accumulate. confocal microscopy revealed that the number of viral cores was severely decreased in vero-ifitm cells when compared to control cells containing the empty vector (fig b) . double-labeled encapsidated viral particles were counted and the ratio of decapsidated viral cores compared to the total number of virions per individual cell was calculated and expressed in percentages (fig a) . the percentage of decapsidated virus severely decreased under ifitm expression. however, ifitm expression produced an accumulation of virions leading to higher numbers of total virions (fig c) . this increase in the number of total virions might be the result of an impaired progression of the asfv replication cycle. these virions would neither proceed with infection nor be degraded and this would be consistent with an inhibition of the membrane fusion capacity at the endosomal level. altogether, these results indicate that viral entry was the rate-limiting step in vero-ifitm and ifitm cells for asfv infectivity. finally, we analyzed the impact of ifitm expression on other infection parameters to find reduced number of copies of asfv genome at hpi (fig a) . ifitm overexpression induced a consistent reduction in infectivity as measured by early protein p expression by flow cytometry (fig b) . however, these reductions were even more marked when analyzing infected cell percentages by late p protein expression (fig c) . finally, we also analyzed viral protein expression by wb (fig d- f) . the expression of protein p at hpi ( fig d) and p at hpi (fig e) resulted significantly reduced as other infection parameters. in the present work, we have studied the antiviral effect of the ifitm family of proteins in the context of cell-adapted asfv infection in vero cells. while different ifitm proteins have been repeatedly described as inhibitors of a broad spectrum of rna viruses [ ] , their antiviral role involving dna viruses is poorly studied and only reported in the rana grylio virus (rgv), blocking the virions at the entry stage into the host cell [ ] . asfv ba v infection in vero cells is significantly affected upon ifn treatment [ , ] . in general, the sensitivity towards the induction of the innate immune response of the host cell has led viruses to acquire different strategies to regulate the ifn pathway for its own benefit. such is the case of asfv viral gene a r, which negatively regulates the induction of ifn through targeting irf in a nfκb-independent manner [ ] . another example is the asfv gene i l, which codifies for a viral tlr homolog with inhibitory activity against ifn [ ] . finally, the myxovirus resistance gene a (mxa), which is another interferon stimulated gene (isg), also inhibits the replication and the late gene expression of asfv [ ] . we report here that upon ifn treatment of vero cells, the distribution of ifitm proteins changes into a perinuclear vesicular pattern resembling endosomes. our analysis of endogenous ifitm expression in the absence of ifn induction showed colocalization with mitochondrial structures. interestingly, ifitm underwent vesicular pattern redistribution around the cell nucleus when overexpressed and upon ifn treatment as well. our characterization of the cellular distribution of ifitm , and also unveiled specific localization patterns linked to the endosomal pathway upon overexpression, particularly in the late endosomal compartments. this distribution is coincident with previously reported data of other groups, which described localization of endogenous ifitm at the plasma membrane and early endosomes, and of ifitm and ifitm in late endosomes and lysosomes [ , ] . interestingly, overexpression of ifitm has been recently described to delay the proteolytic degradation of human papilloma virus (hpv) capsids in keratinocytes [ ] . this, however, did not affect the replication of the virus. our analysis of asfv ba v uncoating correlated the expression of ifitm with a decrease in the numbers of decapsidated asf virions in vero cells. this suggests a possible role for ifitm in inhibiting asfv exit from late endosomes. in contrast, ifitm did not modify the ratio between encapsidated and decapsidated virions. instead, ifitm apparently increased the accumulation of virions that are probably retained and do not proceed to degradation and/ or to a productive infection. these results may also suggest that the presence of ifitm affects the release of the virions from the late endosomal compartments. asfv enters into the host cell by dynamin-dependent and clathrin-mediated endocytosis [ , ] and macropinocytosis [ ] . in fact, endosomal pathway integrity is known to be important for asfv infection, both for culture-adapted isolates in cell lines [ ] or for virulent and attenuated isolates in primary macrophages [ ] . hence, it is not surprising that infection could be impaired by these restriction factors acting at the endosomal membrane. the aforementioned ifitm -and ifitm -mediated inhibition of asfv entry has been previously reported in other viruses, including iav, flaviviruses (denv and wnv) [ ] , filoviruses (marv and ebov) and coronaviruses (such as sars) [ ] . in contrast, infection with alphaviruses, arenaviruses and mlv (a retrovirus) seems to be unaffected by ifitm protein expression [ ] . in general, ifitm-mediated viral inhibition has been related to impaired viralhost membrane fusion subsequent to viral binding and endocytosis [ , ] . ifitm has also been reported to modulate the fluidity and the bending modulus of the cell membrane, thus making it resistant to viral fusion machinery [ ] . we also studied whether the inhibition of asfv entry could be due to an alteration of the endosomal compartments. analysis of endosome distribution upon ifitm overexpression revealed that ifitm and ifitm altered the normal distribution of early and late endosomes and lysosomes. however, this alteration was not found in the presence of ifitm . a collapse of endosomes to the perinuclear area is also a characteristic phenotype of an alteration of endosomal cholesterol efflux [ ] . also, recent publications from our laboratory demonstrated that accumulation of cholesterol in endosomes caused by a chemical inhibition of cholesterol flux, causes virion retention inside endosomes and inhibition of infection progression [ ] . there are currently two proposed models trying to explain the ifitm-mediated inhibition of viral entry. the first one, known as the "tough membrane" model, postulates that intramembranous interactions between adjacent ifitms alter the fluidity and bending of the host cellular membrane, making it resistant to the viral fusion machinery [ ] . the second model suggests that ifitms can induce the accumulation of cholesterol in the endosomal membrane and membrane fusion defects [ ] disturbing intracellular cholesterol homeostasis that finally blocks the viral release into cytosol [ ] . ifitm and ifitm have been previously reported to alter the cholesterol homeostasis at the late endosome, leading to cholesterol accumulation and blocking the viral release [ ] . in the present study, we have described accumulation of cholesterol upon overexpression of ifitm and ifitm . we have recently reported that inhibition of cholesterol exit from endosomes using chemical inhibitors causes retention of virions inside these vesicles, thus impairing progression of the infection [ ] . altogether these data could suggest that the antiviral action of ifitms may affect to a higher extent to those viruses that require the endosomal pathway during the early stages of infection. collectively, our results illustrate a close relationship between the ifitm protein family and the endosomal pathway, leading to the inhibition of asfv infection. the antiviral action of ifitms could rely on alterations of the endosomal physiology and ongoing studies in our laboratory will be focused on antiviral targets at the molecular regulation of late endosomes. also, a role in cell-to-cell viral transmission has been postulated for ifitms, which are incorporated into hiv- virions [ ] . we have performed this study in a cellular system using the cell-adapted ba v isolate in vero cells but these results will be next extended to other asfv isolates with porcine macrophages. further studies will be required for better understanding the relevance of ifitms in the context of asfv infection. in summary, ifitms represent a broad and previously unappreciated class of restriction factors that degrade invading enveloped viruses and may therefore be considered as potential antiviral components to protect the host cell. ifitms restrict the replication of multiple pathogenic viruses transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells molecular analysis of a human interferon-inducible gene family the broad-spectrum antiviral functions of ifit and ifitm proteins the cd /cd signal transducing complex of human b lymphocytes includes the target of antiproliferative antibody- and leu- molecules the ifitm proteins mediate cellular resistance to influenza a h n virus, west nile virus, and dengue virus ifitm is a tight junction protein that inhibits hepatitis c virus entry evidence for paralichthys olivaceus ifitm antiviral effect by impeding viral entry into target cells the antiviral restriction factors ifitm , and do not inhibit infection of human papillomavirus, cytomegalovirus and adenovirus common origin of four diverse families of large eukaryotic dna viruses endosomal maturation, rab gtpase and phosphoinositides in african swine fever virus entry african swine fever virus infects macrophages, the natural host cells, via clathrin-and cholesterol-dependent endocytosis african swine fever virus-cell interactions: from virus entry to cell survival european comission, . . cases of asf confirmed in latvia. the veterinary record epidemiology of african swine fever in poland since the detection of the first case. polish journal of veterinary sciences dynamin-and clathrin-dependent endocytosis in african swine fever virus entry titration of african swine fever (asf) virus. the journal of general virology visualization of the african swine fever virus infection in living cells by incorporation into the virus particle of green fluorescent protein-p membrane protein chimera attenuation of the lysosomal death pathway by lysosomal cholesterol accumulation development of a taq-man pcr assay with internal amplification control for the detection of african swine fever virus the major cellular sterol regulatory pathway is required for andes virus infection the antiviral effector ifitm disrupts intracellular cholesterol homeostasis to block viral entry ifitm restricts influenza a virus entry by blocking the formation of fusion pores following virus-endosome hemifusion alteration of dynein function affects alpha-synuclein degradation via the autophagosome-lysosome pathway phosphatidylinositol -kinases: hostages harnessed to build panviral replication platforms african swine fever virus morphogenesis. virus research effect of interferon-alpha, interferon-gamma and tumour necrosis factor on african swine fever virus replication in porcine monocytes and macrophages. the journal of general virology interferon cures cells lytically and persistently infected with african swine fever virus in vitro identification and utility of innate immune system evasion mechanisms of asfv. virus research a novel tlr inhibitor encoded by african swine fever virus (asfv) inhibition of a large double-stranded dna virus by mxa protein - pmid: ifitm inhibits influenza a virus infection by preventing cytosolic entry distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus african swine fever virus uses macropinocytosis to enter host cells the cd domain of ifitm is required for both ifitm protein association and inhibition of influenza a virus and dengue virus replication cholesterol flux is required for endosomal progression of african swine fever virions during the initial establishment of infection the diverse functions of oxysterol-binding proteins ifitm proteins incorporated into hiv- virions impair viral fusion and spread distance measure software plug-inn of imagej was kindly provided by dr. esteban veiga and giulia morlino from institute for health research "hospital universitario la princesa", cnb, madrid.funding sources for ca: ministerio de economía y competitividad (es) http://www. mineco.gob.es/portal/site/mineco/idi agl - and agl - -r and for ag-s national institute for allergy and infectious diseases (niaid); http://www.niaid.nih.gov/; u ai . key: cord- -synuzaxm authors: borel, nicole; dumrese, claudia; ziegler, urs; schifferli, andrea; kaiser, carmen; pospischil, andreas title: mixed infections with chlamydia and porcine epidemic diarrhea virus - a new in vitro model of chlamydial persistence date: - - journal: bmc microbiol doi: . / - - - sha: doc_id: cord_uid: synuzaxm background: chlamydiae induce persistent infections, which have been associated with a wide range of chronic diseases in humans and animals. mixed infections with chlamydia and porcine epidemic diarrhea virus (pedv) may result in generation of persistent chlamydial infections. to test this hypothesis, an in vitro model of dual infection with cell culture-adapted pedv and chlamydia abortus or chlamydia pecorum in vero cells was established. results: infected cultures were investigated by immunofluorescence (if), transmission electron microscopy (tem) and re-infection experiments. by if, chlamydia-infected cells showed normal inclusions after hpi. dual infections with chlamydia abortus revealed a heterogenous mix of inclusion types including small inclusions consisting of aberrant bodies (abs), medium-sized inclusions consisting of abs and reticulate bodies and normal inclusions. only aberrant inclusions were observable in dual infection experiments with chlamydia pecorum and pedv. tem examinations of mixed infections with chlamydia abortus and chlamydia pecorum revealed aberrant chlamydial inclusions containing reticulate-like, pleomorphic abs, which were up to μm in diameter. no re-differentiation into elementary bodies (ebs) was detected. in re-infection experiments, co-infected cells produced fewer ebs than monoinfected cells. conclusions: in the present study we confirm that pedv co-infection alters the developmental cycle of member species of the family chlamydiaceae, in a similar manner to other well-described persistence induction methods. interestingly, this effect appears to be partially species-specific as chlamydia pecorum appears more sensitive to pedv co-infection than chlamydia abortus, as evidenced by tem and if observations of a homogenous population of aberrant inclusions in pedv - chlamydia pecorum co-infections. chlamydiae are implicated in a wide variety of diseases in both animals and humans. although acute infections in animal chlamydioses are the most commonly reported, chronic chlamydial infections are also associated with a variety of diseases in humans and animals. these latter infections are characterized by inflammation and scarring resulting in significant damage of the host. a causative role in chronic diseases requires that chlamydiae persist within infected tissue for extended periods of time. current theories, based primarily on in vitro data, suggest that chlamydial persistence, and the resulting chronic inflammation, is linked to morphological and metabolic conversion of the actively replicating and intracellular reticulate body (rb) into an alternative, non-replicative form known as an aberrant body (ab) [ ] . in vitro, alterations of the normal developmental cycle of chlamydia trachomatis and chlamydia pneumoniae can be induced by interferon-γ (ifn-γ), tumor necrosis factor-α (tnf-α) and penicillin g exposure as well as amino acid or iron deprivation and monocyte infection [ , ] . to date, in vitro models for animal pathogens, chlamydia abortus and chlamydia pecorum have not been described although both organisms are associated with chronic disease in koalas and small ruminants [ ] . in pigs, several chlamydial species, including chlamydia abortus, chlamydia psittaci, chlamydia pecorum and chlamydia suis, have been implicated in a variety of disease conditions including conjunctivitis, pneumonia, pericarditis, polyserositis, arthritis, abortion and infertility [ ] . in the gastrointestinal tract, chlamydiae appear to be highly prevalent but only occasionally cause enteritis. they have been found in the intestine of diarrheic and healthy pigs and could be demonstrated in mixed enteric infections [ ] [ ] [ ] . pospischil and wood [ ] first described an association between chlamydiaceae and lesions in the intestinal tract of pigs and assumed a synergistic effect in co-existence with salmonella typhimurium. further, mixed infections with eimeria scabra, cryptosporidia, and porcine epidemic diarrhea virus (pedv) have been described in the past. pedv, a member of the family coronaviridae, is a well-known cause of diarrhea in pigs. after the identification of pedv in by pensaert and debouck [ ] , more than a decade passed before the virus could be adapted for propagation in cell cultures. examination of infected vero cell cultures by direct immunofluorescence revealed single cells with granular cytoplasmic fluorescence as well as formation of syncytia with up to - nuclei or more. typical features of syncytial cells were growth, fusion and detachment from cell layers after they had reached a certain size [ ] . biomolecular studies revealed major genomic differences between cell culture-adapted (ca)-pedv and wild type virus [ , ] . cell culture model of co-infection with ca-pedv and chlamydia has been established recently [ ] to investigate the interaction of ca-pedv and chlamydiaceae in mixed infections and to detect possible synergistic or additive effects of possible significance in clinical enteric disease in pigs. in that study, abnormally large chlamydial forms were observed in dually infected cell layers by immunofluorescence suggesting that ca-pedv co-infection might alter the chlamydial developmental cycle in a manner similar to that observed during persistent infections. to confirm these initial observations, we established a cell culture model of mixed infections with chlamydia and a cell culture-adapted porcine epidemic diarrhea virus (ca-pedv) and hypothesized that this would result in the generation of persistent chlamydial forms. this data demonstrates that ca-pedv co-infection, indeed, alters the developmental cycle of chlamydia pecorum and chlamydia abortus in a similar manner to other inducers of chlamydial persistence. vero cells can be co-infected with chlamydia and ca-pedv immunofluorescence (if) labeling was used to investigate the morphologic differences of chlamydia between monoinfected and dually infected monolayers using chlamydia and ca-pedv specific antibodies. control and mock-infected cells did not stain with either antibody. ca-pedv monoinfected cells showed brilliant and distinct, red cytoplasmic fluorescence. syncytia were characterized by accumulation of nuclei in the center or the periphery of the multi-nucleated cells and moderate to bright, fine-granular, cytoplasmic ca-pedv labeling (figure b) . syncytia were categorized into small ( - nuclei), medium ( - nuclei) and large (more than nuclei). in single infection experiments, syncytia at h post infection were mostly large with fewer medium sized syncytia observed (data not shown). numbers of syncytia in ca-pedv single and dual infections were counted on the whole coverslip and mean values were determined. no difference of viral syncytia numbers for ca-pedv monoinfection and dual infection with chlamydia abortus were seen (data not shown). in contrast, numbers of viral synyctia in dual infections with chlamydia pecorum were diminished compared to the respective ca-pedv single infections (table ) . if microscopy of chlamydial single infections revealed intracytoplasmic, mainly round to ovoid, sharply outlined inclusions with brilliant, green fluorescence. chlamydia abortus and chlamydia pecorum infected cells had one to five, finely granular (consisting mainly of ebs) inclusion(s) per cell at h post infection ( figure figure morphology of chlamydia pecorum mono-and coinfection with pedv. a) vero cells were infected with chlamydia pecorum moi for h, with subsequent pedv inoculation and labelled with an anti-chlamydia antibody (green); b) double infected monolayer were labelled for ca-pedv in red, chlamydia in green and dna in blue; c) chlamydia pecorum mono-infected vero cells labelled with an anti-chlamydia antibody (green) and dna staining (blue); d) inclusion size was measured as described and the frequency of chlamydial inclusions assembled into sizes of μm area groups depicted. the difference between mono and double infected monolayers was statistically analyzed using students t-test. the groups were significantly different with p = . . c & a). in general, chlamydial inclusions were smaller and had more variable forms in chlamydia pecorum than in chlamydia abortus single infections. infectivity was almost % and a moderate number of free ebs could be observed. compared to single infections, the size and shape of chlamydial inclusions in pedv co-infections was highly variable. in chlamydia abortus co-infection experiments, three types of inclusions were observed: (i) small inclusions consisting of - aberrant bodies (abs), (ii) medium-sized inclusions consisting of abs and reticulate bodies (rbs), and (iii) large (normal) inclusions consisting of ebs as seen in the single infection experiments (figure b ). in contrast, dual infections with ca-pedv and chlamydia pecorum resulted in the exclusive production of aberrant inclusions containing between - abs. chlamydial inclusions in viral syncytia grew even larger than in non-viral infected vero cells. overall, no normal chlamydial inclusions were observed (figure a & b) . image analysis was used to compare inclusion size in single chlamydiae-infected vero cells with the inclusion size in vero monolayers that subsequently underwent ca-pedv virus infection. to this end, inclusion size was determined in μm and all inclusions were assembled into groups covering μm and multiples of this area. the average frequency of chlamydia pecorum inclusions between μm and μm was significantly reduced when cells were subsequently infected with ca-pedv. in other words, chlamydia pecorum inclusions vero cells were infected with chlamydia abortus with subsequent pedv inoculation and stained as with an anti-chlamydia antibody and dapi; c) frequency of inclusions with various sizes was calculated and mono and double infected cells were compared according to the inclusion size. the difference between mono and double infected monolayers was statistically analyzed using students t-test. the groups were significantly different with p = . . a numbers of syncytia for ca-pedv monoinfection and dual infection with chlamydia pecorum were counted on the whole coverslip. b vero cells were mock infected (mock), c. pecorum infected, ca-pedv infected (ca-pedv) and chlamydia pecorum/ca-pedv co-infected as described. were highly significant smaller in ca-pedv dual infections than in those infections without the addition of virus ( figure d ) as analyzed by t-test (p = . ). the additional changes observed in the shape of all inclusions growing in virus-infected monolayers indicated the induction of chlamydia pecorum persistence, since the finely dispersed staining reverted to grape-like structures (figure a & b ). the changes of chlamydial inclusion size by subsequent virus addition to chlamydia abortus are different to those we observed in the chlamydia pecorum dual infection experiments. the frequency of inclusions observed between a size range of - μm was significantly (p = . ) reduced under virus infection but the amount of medium sized and big inclusions - μm was increased ( figure c ). the morphology of chlamydia abortus inclusions was also found to differ in the population when co-infected with ca-pedv. smaller inclusions were generally observed in aberrant shapes compared to larger inclusions, which appeared similar to normal actively growing inclusions showing finely dispersed staining (figure b ). this effect might be due to an incomplete induction of persistence of chlamydia abortus when cells were ca-pedv coinfected. co-infection with ca-pedv induced ultrastructural morphological changes in chlamydia abortus and chlamydia pecorum persistent forms of chlamydia trachomatis and chlamydia pneumoniae are well described by their characteristic electron microscopic appearance [ , , ] . thus, chlamydial ultrastructure in single and co-infected cells was compared by transmission electron microscopy (tem). at h after viral infection, viral-induced syncytia containing vacuoles filled with viral particles were present in ca-pedv-monoinfected and dual infections. the viral particles showed the typical coronavirus morphology with a diameter between to nm (data not shown). at h after chlamydial infection, large intracytoplasmic chlamydial inclusions in single infected cells could be observed in vero cells infected with chlamydia abortus or chlamydia pecorum. the inclusions observed contained variable numbers of morphologically normal rbs and ebs and were generally located near the host cell nucleus, often surrounded by mitochondria (figure a and b) . tem examinations of mixed infections (ca-pedv and chlamydia abortus or chlamydia pecorum) revealed aberrant chlamydial inclusions containing fewer bacteria than typical inclusions and were located in viral syncytia or single cells without viral infection. aberrant inclusions consisted of reticulate-like, pleomorphic, aberrant bodies (abs), which were in general larger in diameter (up to μm) than typical reticulate bodies (rbs), with a sparse densitometric appearance and no re-differentiation into elementary bodies (ebs). as already observed in if investigations, three types of inclusions were present in dual infections with ca-pedv and chlamydia abortus (figure c ), whereas dual infections with ca-pedv and chlamydia pecorum resulted in the exclusive production of aberrant inclusions consisting of - abs (figure d ). neither chlamydial inclusions nor ca-pedv virions were visible in mock-infected cells. previous studies have demonstrated that chlamydial persistent forms are non-infectious [ ] . reduced number or even a lack of ebs in co-infected cells in tem suggested arrested chlamydial developmental cycle with halted maturation from rb to eb. to ascertain the effect of ca-pedv inhibition of chlamydial eb production, the yield of infective chlamydial progeny was determined after h of re-infection in three independent experiments for chlamydia abortus (figure a ) and for chlamydia pecorum (figure b ). neither mock nor ca-pedv monoinfected cells produced detectable infectious ebs, whereas chlamydia abortus and chlamydia pecorum single infections cells produced abundant ebs. coinfected cells produced fewer infectious ebs than nonviral infected cells, demonstrating that production of infectious chlamydial progeny was essentially diminished by ca-pedv-co-infection. eradication of infectious eb production was almost complete in chlamydia pecorum double infection, analyzed by reinfection experiments and found to be statistically different as analyzed by ttest (p = . ) (figure b ). in chlamydia abortus reinfection analysis, several ebs could still be observed in spite of the co-infection with ca-pedv (figure a this data is consistent with the observations from our if and ultrastructural analysis. chlamydial co-infection does alter ca-pedv infection depending on the chlamydial species but does not alter viral ultrastructure to determine whether chlamydial pre-infection altered subsequent viral infection, numbers of syncytia and ca-pedv-infected cells from single and co-infected monolayers of three unrelated experiments were counted. mock-infected and chlamydia only infected cells produced no virions. the difference between virus-infected cells and co-infection with chlamydia abortus was minimal. the number of syncytia detected were within the same range (data not shown) indicating that chlamydial co-infection with chlamydia abortus does not alter ca-pedv infection or the development of syncytia. in contrast, numbers of syncytia in co-infection with chlamydia pecorum were reduced compared to single ca-pedv infection (table ) . overall numbers of single viral infected cells were low in both single and co-infection experiments, and no significant difference between the two chlamydial species was obvious (data not shown). viral morphology was also studied by tem. in ca-pedv single and co-infected cells, viral particles were unaltered indicating that chlamydial co-infection did not induce any changes in viral ultrastructural morphology. while a previous study [ ] primarily investigated the interaction of ca-pedv and chlamydiaceae in mixed infections to detect possible synergistic or additive effects of these two pathogens, questions remained about whether viral infection could potentially induce the persistent chlamydial phenotype. enlarged chlamydial inclusions were described in that study in the ca-pedv co-infection model with chlamydia abortus and chlamydia pecorum but no further ultrastructural analysis has been subsequently performed. in this study, in vitro models of chlamydia abortus and chlamydia pecorum persistence were established using co-infection with ca-pedv. several experimental methods were used to demonstrate the characteristic features of chlamydial persistence, including altered ultrastructural morphology and decreased production of infectious ebs. our results demonstrated that ca-pedv-co-infection alters the chlamydial developmental cycle similarly to other inducers of chlamydial persistence. a similar co-infection model has been recently described by deka et al. ( ) [ ] . in that study, it was shown that chlamydia trachomatis enters a viable but non-cultivable, persistent state with herpes simplex virus type (hsv- ) co-infected host cells. in contrast, a similar study investigating a coinfection model with chlamydia trachomatis and genital mycoplasmas, mycoplasma genitalium and mycoplasma hominis, did not change the morphology of chlamydial rbs, indicating that co-infection of these two microorganisms is likely to be independent and not related to the onset of chlamydial persistence [ ] . in the study by deka et al. ( ) [ ] , hela monolayers were first infected with chlamydia trachomatis and h later with hsv- . in our study, the optimal experimental protocol for co-infection procedure was developed, based on our own earlier study [ ] , and optimization experiments performed as a part of the current study (data not shown). to this end, vero monolayers were first infected with chlamydia and later with ca-pedv, thus the suspected inducer of persistence would be introduced after chlamydial infection and differentiation into rbs. simultaneous infection of chlamydia and ca-pedv has been performed earlier [ ] , but did not result in persistent infection in our preliminary experiments (data not shown) and was not considered further as interference of chlamydial infection and concurrent viral uptake could have influenced the results. viral infection and subsequent development of syncytia was not affected by co-infection with chlamydia abortus as demonstrated by unaltered numbers of syncytia observed in the coinfection experiments. in contrast, viral syncytia formation was dramatically decreased in vero cells double infected with ca-pedv and chlamydia pecorum. if chlamydia pecorum infection might induce a down regulation of the host pedv receptor needed for syncytium formation at - hours post-chlamydial infection, this could produce a reduction in syncytium formation without reducing viral entry or replication -the possible persistence inducer mechanism. interestingly, chlamydial persistence was more prominent in co-infection with chlamydia pecorum than with chlamydia abortus, indicating possible species-specific differences. limited reports are available for in vitro models of chlamydial persistence from non-chlamydia trachomatis and chlamydia pneumoniae strains. kaltenboeck and storz ( ) [ ] suggested that strain s of chlamydia pecorum is highly nutrient dependent and this could elicit aberrant forms. indeed, aberrant forms of this strain were significantly present in our study. previously, only limited data have been published on persistent infection of l cells with an ovine abortion strain of chlamydia psittaci (current classification: chlamydia abortus) [ ] . it should be noted, that in the latter study, chlamydial persistence was not demonstrated using the characteristic features now associated with the morphology of persistent chlamydial infections. detailed description of electron microscopic observations on the effects of penicillin on the morphology of chlamydia psittaci cal in l cells showing aberrant chlamydial stages were published by matsumoto and manire [ ] . the different occurence of persistent forms in coinfection with chlamydia abortus and chlamydia pecorum, respectively, has not been described before. differences between persistence behaviour are already known (reviewed by hogan et al., ) [ ] not only between different chlamydial species but also between different serovars and strains of chlamydia pneumoniae and chlamydia trachomatis, respectively. the fact that chlamydia pecorum strain s is an original swine isolate whereas chlamydia abortus strain s / originates from a sheep abortion and, thus, from another animal species could have an impact but needs further investigation. the mechanism by which ca-pedv interferes with chlamydial developmental cycle and chlamydial persistence is still unclear. it is known that vero cells, a monkey kidney epithelial cell line, is deficient for interferon production [ ] ; thus, this cytokine group well known to be capable of inducing in vitro persistence in chlamydia pneumoniae [ ] , cannot be relevant for our co-infection persistence model. co-infection experiments with ca-pedv are best performed with vero cells, as they have been shown to be permissive for viral replication in contrast to other cell lines such as pd , pk , and hrt cell lines [ ] . specific measurements of primate cytokines in our co-infection model are planned in the future to elucidate the mechanism leading to chlamydial persistence. the herpes simplex virus (hsv) co-induced chlamydia trachomatis persistence model [ ] has been recently been shown not to be mediated by any known persistence inducer or anti-chlamydial pathway recently [ , ] . instead, it was hypothesized by the authors that hsv- attachment and/or entry into the host cell is sufficient for stimulating chlamydial persistence, suggesting a potential novel host signaling pathway could be responsible for inducing chlamydial persistence. a very recent publication by the same group showed that hsv replication is not necessary for persistence induction and that chlamydial activity could be recovered after coinfection with uv-inactivated hsv- . finally, it was concluded that the interaction of hsv glycoprotein d with the host cell surface is crucial to trigger chlamydial persistence [ ] . female genital tract infection often has a complex etiology, where chlamydia trachomatis is present together with one or more genital agents. epidemiological and clinical studies have shown that double infection with hsv- and chlamydia trachomatis occurs in vivo; thus, the in vitro model described by deka et al. ( ) [ ] represents a realistic situation in human medicine. similarities exist to the in vitro model established in this study as simultaneous intestinal infection with different pathogens is possible in swine in vivo. a recent study [ ] documented the occurrence of aberrant chlamydial bodies in vivo in intestinal tissues of pigs. in this study, aberrant bodies of chlamydia suis were demonstrated and characterized in the gut of pigs experimentally infected with salmonella typhimurium by transmission electron microscopy. it was concluded by pospischil et al. [ ] that aberrant bodies occur in vivo in pigs and that the gnotobiotic pig model might be suitable for the study of chlamydial persistence in vivo. available intestinal tissues from experimentally infected gnotobiotic piglets (single infection and coinfection with chlamydia and ca-pedv, respectively) will be investigated in the future with the aim of further characterization of abs in vivo. although chronic chlamydial diseases in animals are of economic impact, the pig model may also reveal the important link between persistence in vitro and in vivo and, thus, help to elucidate mechanisms of chronic human chlamydial infections in the future. the present study reports a new persistence model of chlamydia in co-infection with porcine epidemic diarrhea virus (pedv). pedv-co-infection altered the chlamydial developmental cycle similarly to other known inducers of chlamydial persistence. this new animal model could provide the important link between persistence in vitro and in vivo and, thus, would help to elucidate mechanisms of chronic human chlamydial infections in the future. growth medium (gm) for normal cell propagation was minimal essential medium (mem) with earle's salts, mm hepes, without l-glutamine (gibco, invitrogen, carlsbad, ca) and supplemented with % fetal calf serum (fcs) (bioconcept, allschwil, switzerland), mm glutamax-i ( mm, gibco) and . mg/ml gentamycin ( mg/ml, gibco). gm without gentamycin was used for the propagation of cells for infection experiments. infection medium was prepared as gm but without gentamycin and fcs, and was used for the infection and for the h incubation period after the infection with ca-pedv, respectively. incubation medium was prepared as gm without gentamycin, freshly supplemented with μg/ml cycloheximide (sigma, buchs sg, switzerland), and used after an infection for estimation of the chlamydial titer (ifu determination). vero cells (african green monkey kidney cells, crl american type culture collection) were seeded on round plastic coverslips ( mm diameter, bibby sterilin, stone, uk) and cultured in gm without gentamycin at °c until they reached confluence. before inoculation, the cells were washed once with phosphate buffered saline (pbs). two different chlamydial strains of chlamydiaceae were used in this study: chlamydia abortus s / (ovine abortion strain, kindly donated by dr. g.e. jones, moredun research institute, edinburgh, gb) and chlamydia pecorum s (intestinal swine isolate, kindly provided by prof. j. storz, baton rouge, louisiana, la, usa). for initial culturing, chlamydial strains were cultured in embryonated chicken eggs, and yolk sac material was harvested, diluted : in sucrose-phosphate-glutamate (spg) medium and stored at - °c. yolk sac-derived chlamydiae were then propagated in hep- cell (atcc ccl- ) monolayers and elementary bodies (ebs) were harvested and purified by disruption of hep- cell monolayers with a cell scraper, sonication and centrifugation over a renografin density gradient as described elsewhere [ ] . eb suspensions were stored in sucrosephosphate-glutamic acid buffer at - °c, after which viable titers were established using standard methods. moi of was used for chlamydial monoinfection and mixed infection, respectively. ca-pedv strain cv (kindly provided by prof. dr. m. ackermann, institute of virology, university of zurich) was propagated as previously described [ ] . the virus ( , tcid /ml) was used undiluted ( ml , tcid /ml). vero cells, an african green monkey kidney cell line (atcc crl ), were used for all infection experiments. they were propagated in gm without gentamycin at °c in an atmosphere of % co . vero cells were divided into four groups: for mock infection, chlamydial infection, ca-pedv infection, and both chlamydia and ca-pedv double infection. host cells were infected with a moi of for chlamydia and an infective dose of × , tcid /ml for ca-pedv, respectively. for ca-pedv monoinfections and negative controls, infection medium was used. all co-infection experiments were done three times and monoinfections with chlamydia and ca-pedv were performed simultaneously. the optimal experimental protocol (adding the virus several hours after chlamydial infection) for co-infection procedure was developed before (data not shown). for dual infections, cell monolayers were first infected with chlamydia at a moi of . all coverslips were centrifuged at × g for h at °c. timepoint (t ) was defined after centrifugation and supernatant was replaced subsequently by incubation medium. infected monolayers were then incubated for h at °c (t -t ). all cell layers for dual infections or ca-pedv monoinfection were exposed to a ca-pedv suspension ( × , tcid ), the samples were centrifuged again for × g for h at °c and incubated for h at °c. after this incubation period, all monolayers were fixed and further investigated by indirect immunofluorescence and transmission electron microscopy. re-infection experiments were performed to compare the production of infectious chlamydial elementary bodies (ebs) between monoinfections and mixed infections. for indirect immunofluorescence analyses, infected cells were fixed in absolute methanol (- °c) for min. and if labeling of cell cultures was performed immediately after fixation. for viral antigen detection, a mouse monoclonal antibody against the m protein of pedv (mcab , kindly provided by prof. dr. m. ackermann, institute of virology, university of zurich), diluted : in pbs supplemented with bsa, and an alexa fluor -conjugated secondary antibody (goat anti-mouse, : , molecular probes, eugene, usa) were used. chlamydial inclusions were labeled with a chlamydiaceae family-specific mouse monoclonal antibody directed against the chlamydial lipopolysaccharide (mlps; clone aci-p, progen, heidelberg, germany) and a secondary alexa fluor -conjugated secondary antibody (goat anti-mouse, : , molecular probes). dna was labeled with μg/ml ', -diamidin- '-phenylindoldihydrochlorid (dapi, molecular probes). all staining procedures were conducted at room temperature. antibody incubations were carried out for h (primary antibodies) or min (secondary antibodies), respectively, with three washes of pbs following fixation, between and after applications of the different antibodies. dually infected cell layers were stained using sequential double immunofluorescence labeling. uninfected vero cells were used as a negative control. coverslips were mounted with immumount (shandon, pittsburgh, usa) on glass slides and investigated using a leica fluorescence microscope. coverslips from all experimental conditions were fixed in . % glutaraldehyde (electron microscopy sciences, ft. washington, usa) for - h, and processed by routine methods for embedding in epoxy resin (fluka). appropriate areas for ultrastructural investigation were selected using semithin sections ( μm) stained with toluidine blue (fluka, buchs sg, switzerland). ultrathin sections ( nm) were mounted on gold grids (merck eurolab ag, dietlikon, switzerland), contrasted with uranyl acetate dihydrate (fluka) and lead citrate (lead nitrate and tri-natrium dihydrate; merck eurolab ag) and investigated in a philips cm electron microscope. at h after chlamydial infection, monolayers were scraped into ml of cold infection medium, pelleted and resuspended in ml of fresh medium. infected host cells were lysed by sonication and centrifuged ( g for min) to remove pellet cell debris. supernatants were centrifuged once ( , g for min). final eb pellets were resuspended in μl of spg and used to infect vero cells plated on glass coverslips in duplicate in dilution series. all coverslips were centrifuged at × g for h at °c. after centrifugation, the vero cells were refed with medium containing μg/ml cycloheximide and subsequently incubated for h at °c. fixation and staining of chlamydia, ca-pedv and dna was performed as described above. the number of inclusions in random microscopic fields per sample was determined using a leica fluorescence microscope at a magnification of ×. duplicate coverslips were counted and the counts were averaged. the number of inclusion-forming units (ifu) in the indiluted inoculum was then calculated and expressed as ifu per cells as described by deka et al., [ ] . from duplicate samples of three independent experiments uniform random sampled images were acquired using a widefield microscope (leica lx, leica microsystems mannheim, germany). cells and inclusions were automatically detected according to size, shape and intensity and counted using imaris (bitplane ag, zürich switzerland). chlamydial persistence: beyond the biphasic paradigm persistent chlamydiae: from cell culture to a paradigm for chlamydial pathogenesis morphologic and antigenic characterization of interferon gamma-mediated persistent chlamydia trachomatis infection in vitro prevalence of intestinal chlamydial infection in pigs in the midwest, as determined by immunoperoxidase staining intestinal chlamydia in finishing pigs intestinal chlamydia in pigs a new coronavirus-like particle associated with diarrhea in swine propagation of the virus of porcine epidemic diarrhea in cell culture sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic orf pedv leader sequence and junction sites mixed infections in vitro with different chlamydiaceae strains and a cell culture adapted porcine epidemic diarrhea virus electron microscopic observations on the effects of penicillin on the morphology of chlamydia psittaci chlamydia pneumoniae expresses genes required for dna replication but not cytokinesis during persistent infection of hep- cells chlamydia trachomatis enters a viable but non-cultivable (persistent) state within herpes simplex virus type (hsv- ) co-infected host cells chlamydia trachomatis and genital mycoplasmas in the co-infection model -in vitro study biological properties and genetic analysis of the omp a locus in chlamydiae isolated from swine persistent infection of l cells with an ovine abortion strain of chlamydia psittaci characterization of the interferon regulatory factor -mediated antiviral response in a cell line deficient for ifn production an early event in the herpes simplex virus type- replication cycle is sufficient to induce chlamydia trachomatis persistence herpes simplex virus co-infection-induced chlamydia trachomatis persistence is not mediated by any known persistence inducer or anti-chlamydial pathway interaction of hsv- glycoprotein d with the host cell surface is sufficient to induce chlamydia trachomatis persistence aberrant chlamydial developmental stages in the gastrointestinal tract of pigs spontaneously and experimentally infected with chlamydia suis purification on renografin density gradients of chlamydia trachomatis grown in the yolk sac of eggs submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors would like to thank lisbeth nufer of the laboratory staff at the institute of veterinary pathology, zurich, for her excellent technical assistance. we would also like to thank dr. monika engels and eva loepfe, institute of virology (head: prof. m. ackermann), vetsuisse faculty, university of zurich for providing the porcine epidemic diarrhea virus. we thank dr. adam polkinghorne, queensland university of technology, brisbane, australia, for manuscript editing. this work was supported by a grant from the university of zurich (forschungskredit).author details institute of veterinary pathology, vetsuisse faculty, university of zurich, zurich, switzerland. center for microscopy and image analysis, university of zurich, zurich, switzerland. authors' contributions nb conceived of the study, planned the experiments, and drafted the manuscript. cd and uz performed the imaging and statistical analyses. as and ck carried out the cell culture experiments including immunofluorescence and transmission electron microscopy. ap participated in the design and coordination of the study and helped to draft the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- - yq zj authors: klingström, jonas; Åkerström, sara; hardestam, jonas; stoltz, malin; simon, melinda; falk, kerstin i.; mirazimi, ali; rottenberg, martin; lundkvist, Åke title: nitric oxide and peroxynitrite have different antiviral effects against hantavirus replication and free mature virions date: - - journal: eur j immunol doi: . /eji. sha: doc_id: cord_uid: yq zj reactive nitrogen intermediates (rni), like nitric oxide (no) and peroxynitrite, have antiviral effects against certain viruses. hantaviruses, like other members of the bunyaviridae family, have previously not been shown to be sensitive to rni. in this study, we compared the effects of no and peroxynitrite on hantavirus replication and free mature virions in vitro, and of inducible nitric oxide synthase (inos) in hantavirus‐infected suckling mice. the no‐generating compound s‐nitroso‐n‐acetylpenicillamine (snap), as well as cytokine‐induced no, strongly inhibited hantavirus replication in vero e cells, while pretreatment of free virions with snap only had a limited effect on their viability. in contrast, ‐morpholinosydnonimine hydrochloride (sin‐ ), a peroxynitrite donor, inhibited virus replication only to a very low extent in vitro, but pretreatment of virus with sin‐ led to a considerably lowered viability of the virions. infections of various human cell types per se did not induce no production. the viral titers in inos(–/–) mice were higher compared to the controls, suggesting that no inhibits hantavirus replication in vivo. in conclusion, we show that no has strong antiviral effects on hantavirus replication, and peryoxynitrite on mature free virions, suggesting that different rni can have different effects on various parts of the replication cycle for the same virus. hantaviruses cause two severe and often fatal human zoonotic diseases, hemorrhagic fever with renal syndrome (hfrs) in the old world and hantavirus cardiopulmonary syndrome (hcps) in the new world. hantaviruses, belonging to the bunyaviridae family, have a negative sense tripartite rna genome encoding four structural proteins: the nucleocapsid (n) protein, two glycoproteins, and an rna-dependent rna polymerase [ ] . the natural hosts are rodents, and the virus is transmitted to humans via inhaled contaminated rodent excreta. in contrast to human infections, the natural rodent hosts do not show any symptoms after infection [ ] . the pathogenesis in man is only poorly understood, but immune-mediated mechanisms have been suggested [ , ] . nitric oxide (no), a gaseous free radical, is an important molecule playing a key role in a wide range of biological processes, such as vasomotor tone regulation, neurotransmission, and immune responses. no inhibits the replication of certain dna and rna viruses, for instance poliovirus, japanese encephalitis virus, mouse hepatitis virus, vesicular stomatitis virus, herpes simplex virus type , vaccinia virus, epstein-barr virus, influenza virus and sars coronavirus [ ] [ ] [ ] . however, the possible antiviral effect of no and peroxynitrite on hantaviruses, or other viruses within the bunyaviridae family, has previously not been reported. during inflammation, no and superoxide (o -) together form peroxynitrite (onoo -), and other reactive nitrogen intermediates (rni) [ ] . recently, it was shown that peroxynitrite has antiviral capacities both against coxsackievirus replication and free virions, suggesting that also other viruses might be sensitive to peroxynitrite [ ] . although no and peroxynitrite can inhibit viral replication, and thereby contribute to the clearance of virus from the circulation, highly elevated levels of rni during disease can be deleterious [ , ] , due to oxidation and nitration of cellular lipids, dna and proteins [ ] . elevated levels of nitrate/nitrite, stable end-products of no, have been found in hiv-infected individuals, and no has been suggested to play a role in the pathogenesis of aids [ ] . influenza virus pneumonia [ ] and neurotropic virus infections are other diseases where no is believed to contribute to the pathogenesis [ ] , and we have detected elevated levels of no production in suckling mice that succumbed to hantavirus infection [ ] . on the other hand, inducible nitric oxide synthase (inos) deficiency had no impact on the pathology in vaccinia virus and corona virus infections of mice [ ] , showing that no-induced pathology is not a general feature during virus infections. stable end-products of rni have been found at elevated levels in both hfrs and hcps patients [ ] [ ] [ ] , as well as in monkeys infected with puumala hantavirus (puuv) [ ] . in contrast, infection of the natural host peromyscus maniculatus with sin nombre hantavirus (snv) does not induce no production [ ] . the elevated levels of rni detected in hfrs/hcps patients have been suggested to play a part in the pathogenesis [ ] . a variety of cell types and tissues generate no through the conversion of l-arginine into l-citrulline through three distinct isoforms of the enzyme nitric oxide synthase (nos) [ ] . two forms of nos, neuronal nos (nnos or nos ) and endothelial nos (enos or nos ) are constitutively expressed, whereas inos (or nos ) is strongly induced by cytokines and other immunoregulatory stimuli [ ] . in the present study, we investigated the effect of no and peroxynitrite on hantavirus replication in vero e cells and on free mature virions by using s-nitroso-nacetylpenicillamine (snap; an no-donor), -morpholinosydnonimine hydrochloride (sin- ; a peroxynitrite donor), and by stimulating endogenous no production by inos with cytokines. furthermore, the effect on no production by hantavirus infection of several different types of cells in vitro was measured to test if hantavirus infection per se could induce no production, and inos -/suckling mice were infected to test if inos is a part of the antiviral response against hantaviruses, and/or the pathogenesis, in suckling mice. in a first set of experiments, we tested if hantaan hantavirus (htnv) infection of different cell lines induced no production. cells were infected with multiplicity of infection (moi) of htnv and then incubated without change of media. elevated levels of nitrite were detected neither in the supernatants from the human lung epithelial cell lines hl and a , the monkey kidney epithelial cell lines vero and vero e days post infection, from the human hepatoma cell line huh- days post infection, nor from the human cervix epithelial cell line hela, a primary culture of human umbilical vein endothelial cells (huvec) or the human monocytic cell line monomac days post infection. the viability of htnv-infected cells treated with lm snap or lm of the control n-acetylpenicillamine (nap), or medium alone, starting h before infection and then replenished every h, was examined. no toxicity of snap could be measured using an mtt test h post infection (data not shown), showing that lm snap is not toxic for vero e cells. the nitrite concentration in medium measured h after snap treatment was approximately half of the concentration of snap added to the medium, clearly showing that no was released by snap under these conditions (fig. ) . as expected, no nitrate was detected in medium from cells incubated with nap for h (fig. ) . whether no had an effect on htnv replication was then tested. htnv-infected cells were incubated with . - lm snap or nap, starting h after infection. the media were subsequently changed every h, and supernatants were drawn at h post infection and titrated. no viable viruses were detected in supernatants from cells treated with lm snap ( fig. a) . from cells treated with lm snap, % less viable virus was obtained, whereas lower concentrations of snap had no detectable effect on the virus replication, as compared to nap-treated or medium controls ( fig. a , and data not shown). to test if the replication of other hantaviruses was also sensitive to snap, cells were infected with puuv, dobrava hantavirus (dobv) and saaremaa hantavirus (saav), followed by treatment with lm snap, lm nap, or medium alone from h post infection. a complete inhibition of released virus was observed for puuv at h post infection, and for dobv at h post infection, whereas an approximately % inhibition was detected for saav at h post infection (fig. b ). since it has been shown that replication of vesicular stomatitis virus is more efficiently inhibited when the cells are pretreated with exogenous no donors [ ] , we compared treatment of vero e cells with lm snap starting h before, h after, or h after htnv infection. the media were changed every h, and supernatants were sampled h post infection. no difference was observed between adding snap h before infection or h post infection (fig. c) , showing that pretreatment is not needed for no-induced inhibition of replication. however, a stronger inhibition was observed when snap was added h before or h post infection, as compared to h post infection to investigate if no could inhibit hantavirus replication in an already established infection, we infected cells with htnv and incubated them for wk (at this time point all cells were infected; data not shown). cells were then treated with . - lm snap. at h after the initial treatment with and lm snap, the titers of released virus were approximately and . % lower, respectively, as compared to cells treated with nap or medium alone (fig. d ). lower concentrations of snap had no effect on the virus titers (fig. d , and data not shown). we have previously shown that il- b together with ifn-c up-regulates inos expression in vero e cells [ ] . here, we further examined the effect of cytokineinduced no on hantavirus replication in vitro. treatment of cells with il- b ( ng/ml) alone had no effect on htnv replication (data not shown), whereas ifn-c ( u/ml), as reported earlier [ ] , inhibited the viral replication (data not shown). no increased level of nitrite was detected in cells treated with il- b or ifn-c alone (data not shown), suggesting that the expression of inos in vero e cells requires both il- b and ifn-c. approximately lm of nitrite was detected in the medium h after stimulation with il- b and ifn-c. to test the effect of cytokine-induced no on htnv replication, cells were infected with htnv. after h, cells were stimulated with il- b and/or ifn-c in the presence of mm of the nos inhibitor n g -monomethyl-l-arginine (l-nmma) or the control n g -monomethyl-darginine (d-nmma). l-nmma and d-nmma were subsequently added to the media also h post infection. supernatants were collected h post infec-tion for virus titration. the viral titers in supernatants from cells stimulated by il- b combined with l-nmma or d-nmma showed no clear differences (approximately % more viruses in l-nmma + il- b-treated cells compared to d-nmma + il- b-treated cells). similarly, l-nmma or d-nmma had no clear effect on the viral titers from cells stimulated by ifn-c (approximately % more viruses in d-nmma + ifn-c-treated cells compared to l-nmma + ifn-c-treated cells), showing that the inhibition of virus replication by ifn-c alone is no independent. in contrast, approximately % higher htnv titers were observed in supernatants from cells treated with the nos inhibitor l-nmma, as compared to supernatants from cells treated with the control d-nmma, in cells incubated in the presence of il- b together with ifn-c (fig. ) . thus, cytokine-induced no can inhibit htnv replication in vero e cells. we further tested if no affected the levels of n protein expressed after infection of vero e cells. cells were treated with lm snap, lm nap, or medium alone, with start at h post infection, and then media were changed every h. treatment with lm snap had no effect on the total cellular protein, or b-actin, levels in vero e cells ( [ ] , and data not shown). in samples drawn h post infection, htnv n protein was detected in nap-treated and untreated cells, but not in snap-treated cells (fig. a) . we then tested if no also had an effect on viral rna by performing real-time pcr on the puuv s-segment. cells were infected with puuv and treated with lm snap, lm nap or normal medium, as described above. the levels of viral rna in the cells h post infection were analyzed. approximately % less viral rna was detected in snap-treated cells as compared to the nap-or medium-treated cells (fig. b) . as peroxynitrite was recently shown to have antiviral capacities [ ] , we then investigated if hantavirus replication was sensitive to treatment with peroxynitrite. in medium with lm sin- , lm nitrite was detected h after incubation, showing that most of the added sin- had decayed (data not shown). to vero e cells, lm sin- was added after infection with htnv, and fresh medium containing sin- was added at h and h after infection. at h post infection, the supernatant was collected and titrated. approximately % less viable virus were observed in cells incubated with lm sin- , as compared to controls (fig. ) . to test if no or peroxynitrite had a direct inhibitory effect on free mature virions, htnv was incubated with lm to mm snap, lm to mm sin- , mm nap, or with normal medium for days at + c before titration. the levels of nitrite detected in the medium at this time point corresponded to approximately half of the added concentration of snap and sin- (data not shown). no nitrite was detected after addition of mm nap to the media (data not shown). the virus was fold diluted before titration to rule out the effect of potential inhibition of viral replication by residual snap or sin- . while lm sin- inactivated approximately % of the virus, lm sin- inactivated more than % of the virus, and mm sin- almost all (fig. ). approximately % reduction in viability was observed for mm and lm snap, as compared to the nap or medium controls (fig. ) . to test if no has a role in the antiviral defense in vivo, we infected suckling c bl/ mice (inos -/and inos +/+ controls) with focus forming units (ffu) dobv, previously shown to induce no production in and to be lethal for suckling mice [ ] . all mice in the two groups (inos -/-, n = ; controls, n = ) showed ruffled fur, paralysis of the limbs, and progressively diminishing mobility, at day after infection, and were sacrificed at higher levels of dobv were observed in the inos -/-( ae ffu/g brain) compared to the control ( ae ffu/g brain) mice (fig. ) , suggesting that no produced by inos has antiviral properties against hantaviruses in vivo. the major finding in this study is that different rni can have different effects on various parts of the replication cycle for viruses; no showed a strong antiviral effect on the hantavirus replication in vitro but only a minor effect on free viruses, while the opposite was observed for peroxynitrite. furthermore, we showed for the first time that a member within the bunyaviridae family is sensitive to no and peroxynitrite. it should be noted that although snap releases no into the medium, some no might escape into the atmosphere, and furthermore, a portion of the no radicals produced most probably oxidizes into various reaction products of no Á , such as nonoates, s-nitrosothiols, nitrite, and nitrous acid that could account for parts of the activity. no will also react with o in the medium and form peroxynitrite. and although most of the no and o produced by sin- will immediately form peroxynitrite, some no will be produced that might not react with o -. superoxide can also form hydrogen peroxide, which in turn can form hypochlorus acid and other oxidants. these reactive nitrogen intermediates and reactive oxygen intermediates can penetrate cellular membranes and react with pathogen targets. thus, the effect we have observed might be even more polarized, as some of the effect of snap observed on viable free virions might be explained by the formation of small amounts of peroxynitrite and other nitrogen intermediates, and the minor antiviral effect of sin- observed on hantavirus replication in vitro might partly be explained by the production of no and other intermediates. the half-life of no and peroxynitrite, endogenously produced or formed after decomposition of snap and sin- , is very short, and it is therefore difficult to adequately measure the amounts of no or peroxynitrite at a given time point. the levels of nitrate/nitrite observed in patients indicate the accumulated levels of no and/or peroxynitrite, but say little about the concentration of them at a certain time point. it is therefore not possible to state that the amount of no and peroxynitrite formed in vitro by snap and sin- , respectively, and shown to be antiviral against hantaviruses, are physiologically relevant. however, the finding that inos -/suckling mice had higher levels of replicating virus than controls is in line with the finding that cytokine-stimulated no production inhibited hantavirus replication in vero e cells. furthermore, davis and coworkers recently reported clearly elevated levels of nitrate/nitrite in hcps patients [ ] , and groeneveld and coworkers showed the same for hfrs patients [ ] . thus, our results suggest that the levels of no and peroxynitrite formed in patients might reduce hantavirus replication and/or damage free virions. not all the mechanisms behind the antiviral effect of rni are known. however, at least three different mechanisms are known for no and one for peroxynitrite: (i) s-nitrosylation of cysteine residues of viral proteins needed for replication; for instance, the inhibition of coxsackievirus replication is related to s-nitrosylation of cysteine protease b [ ] . (ii) enhanced mutation rate: no has been shown to enhance the mutation rate of another rna virus, the sendai virus [ ] . (iii) s-nitrosylation of host cellular proteins needed for virus replication: the antiviral effect of no against some viruses depends on pretreatment of cells with no before infection [ , ] . peroxynitrite has been reported to inhibit coxsackievirus rna entry into host cells [ ] . the mechanisms behind the antiviral effect of no and peroxynitrite on hantavirus replication and free virions, respectively, remain to be studied. the finding that pretreatment of cells with snap was not needed for inhibition of the virus replication suggests that s-nitrosylation of host cellular proteins is not instrumental for no-mediated inhibition of hantavirus replication. furthermore, it seems likely that no and peroxynitrite have different targets, as they only show a minor overlap in their potential to interfere with replication and to inactivate free virions. inos is the major source of no during virus infection. there are two pathways for inos induction during infections: direct up-regulation by the virus or indirect up-regulation via cytokine-dependent mechanisms [ ] . direct up-regulation by virus has been shown for respiratory syncytial virus, human immunodeficiency virus, and hepatitis c virus infection [ ] [ ] [ ] [ ] . the exact mechanism(s) leading to elevated levels of rni during human hantavirus infection are currently not known, but the finding that infection of cells in vitro did not induce detectable levels of nitrite suggests that rni are produced as a response to the elevated levels of cytokines, like tnf-a and ifn-c, detected in patients [ ] . rni has been suggested to be involved in hantavirus pathogenesis [ ] . elevated levels of no have been detected in man [ ] [ ] [ ] and monkeys [ ] , in whom hantavirus infections induce clinical symptoms, but are normally cleared within weeks after infection. we previously showed that dobv, but not saav, was lethal for suckling mice and that increased levels of no production were detected in lethally infected mice [ ] . furthermore, we also observed replicating virus in saav-inoculated mice days after infection, whereas mice that survived dobv infection had no replicating viruses in the brain [ ] . together with the finding that inos -/suckling mice showed higher titers of replicating virus in the brain compared to normal c bl/ mice and that both strains showed severe symptoms at the same day after infection, the results might indicate that no, at least in mice, is more likely to be involved in viral clearance than in pathogenesis. in conclusion, we report that no and peroxynitrite, two rni, both have antiviral effects on hantaviruses, and that this effect is caused by inhibition of viral replication by no at an early step in infection, and by direct inactivation of free virions by peroxynitrite. furthermore, our results strengthens the suggestion that peroxynitrite is an endogenous effector of the antiviral immune response [ ] . the viruses used were the vero e cell line-adapted htnv, strain - [ ] , dobv, strain slovenia [ ] , saav [ ] , and puuv, strain kazan e [ ] . propagation and titration of the viruses were performed on vero e cells [vero c ; american type culture collection (atcc), manassas, va], as described [ ] . the cells used were a , hela, hl, huh- , huvec (clonetics, biowhittaker, walkersville, wv), monomac (kindly provided by sa björndal, swedish institute for infectious disease control, solna, sweden), vero and vero e . a , hela, hl, huh- , vero, and vero e cells were grown in emem supplemented with % fcs, u/ml penicillin, lg/ml streptomycin, and . g/l bicarbonate (sigma, st. louis, mo), huvec in egm- -mv medium supplemented with egm- -mv singlequots (clonetics), and monomac in rpmi supplemented with % fcs, u/ml penicillin and lg/ml streptomycin. suckling c bl/ and c bl/ inos -/mice, were purchased from mtc, breeding unit, karolinska institutet, stockholm, sweden. suckling mice were inoculated intracerebrally with ll dobv. infected mice were kept in biological safety isolators. after sacrifice, hearts and brains were removed aseptically; brains were minced in pbs and stored at - c until further use. hearts were stored at - c with pbs for h; after thawing, the supernatant was transferred to a new tube and used for the detection of hantavirus-specific antibodies [ ] . the care of all animals used in the present study was in compliance with the relevant guidelines and requirements of the swedish institute for infectious disease control, stockholm, sweden. recombinant human il- b and ifn-c were purchased from peprotech (london, uk). sin- , snap and nap, were obtained from sigma. l-nmma and d-nmma were purchased from calbiochem (la jolla, ca). samples were diluted tenfold in hbss supplemented with % hepes, % fcs, u/ml penicillin and lg/ml streptomycin, and incubated on confluent vero e cells in -well plates. after h of incubation, cells were overlaid with . % agarose-medium and incubated for a further - days, depending on the virus, at c, % co . foci of infected cells were stained with polyclonal rabbit anti-htnv or rabbit anti-puuv sera, followed by horseradish peroxidase (hrp)conjugated goat anti-rabbit igg (bio-rad, hercules, ca) and were visualized with , , , -tetramethylbenzidin (sigma) and counted [ ] . confluent vero e cells grown on -well plates were washed, and medium alone or medium containing sin- , snap or nap was added. cells were infected with ffu of hantavirus, corresponding to . moi. media, with or without chemicals, were changed every h. supernatants were collected h after the last treatment and were subsequently titrated on vero e cells as described above. essentially the same protocol was used for endogenously produced no [ ] : ng/ml il- b and/or u/ml ifn-c was added to the media h after virus infection. l-nmma ( mm), a general nos inhibitor, or d-nmma as control for l-nmma, was added to the cells at and h after virus infection. supernatants were collected for virus titration h post infection. an mtt [ -( , -dimethylthiazol- -yl)- , -diphenyl- h-tetrazolium bromide] assay was used to measure mitochondrial function, which served as an index of viable cells, in the snaptreated, nap-treated and untreated cells. the mtt cell proliferation assay was carried out according to the manufacturer's instructions (atcc). no rapidly reacts with oxygen to form nitrite and nitrate, its two stable end-products [ ] . production of no in vitro, and release of no from snap and of peroxynitrite from sin- , was measured indirectly in cell culture supernatants by determination of the level of nitrite using the griess assay. supernatant samples, and sodium nitrite as standard, were mixed with equal volumes of griess reagents ( % sulfanilamide and . % naphtylethylenediamide, in % phosphoric acid), and the optical density at nm was measured by spectrophotometry. the nitrite standard was diluted in the same medium as used for the samples. vero e cells were infected with htnv and treated with snap, nap or medium alone as described above. at the end of infection, cells were collected and homogenized in lysis buffer ( mm tris-hcl, mm nacl, mm edta, mm naf, mm na vo , % triton x- , mm phenylmethylsulfonyl fluoride, lg/ml aprotinin and leupeptin). lysates were mixed : in sample buffer ( mm tris-hcl, ph . , . % sds, % glycerol, % b-mercaptoethanol and bromophenol blue) resolved in % tris-glycine polyacrylamide gels and transferred to nitrocellulose membranes. blocking was performed overnight at c in % nonfat dry milk in . % tween- in pbs. the membranes were subsequently incubated with hantavirus n-specific mab c [ ] and b-actin-specific mab for h at room temperature, followed by hrp-conjugated secondary antibodies. proteins were detected with ecl plus western blotting detection kit (amersham biosciences, uppsala, sweden). rna was extracted from puuv-infected cells using tripure (roche diagnostics, lewes, uk), according to the manufacturer's instructions. first-strand cdna synthesis (amersham pharmacia biotech inc., piscataway, nj) was performed according to the manufacturer's instructions with primer pd(n) . taqman real-time pcr was performed with nm of primer f -gtgcaccagatcggtgtcc- , nm of primer r -caattcagccatcccagca- and nm of taqman mgb probe t -cctacatgcatttatg- on a ht sequence detection system (applied biosystems, foster city, ca) with software sds version . [ ] . rna extracted from stocks of puuv kazan-e with known concentrations of virus (measured as ffu on vero e cells) was used as a standard. hantaviruses: genome structure, expression and evolution hantaviruses: a global disease problem clinical aspects of nephropathia epidemica (puumala virus infection) in europe: a review. scand does nitric oxide play a critical role in viral infections? inhibition of influenza virus replication by nitric oxide nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus specificity of a third kind: reactive oxygen and nitrogen intermediates in cell signaling peroxynitrite inhibition of coxsackievirus infection by prevention of viral rna entry nitric oxide and virus infection nitric oxide synthesis in patients with advanced hiv infection rapid interferon gamma-dependent clearance of influenza a virus and protection from consolidating pneumonitis in nitric oxide synthase -deficient mice but not saaremaa, hantavirus is lethal and induces nitric oxide production in suckling mice il- and il- antagonize il- -mediated protection against acute vaccinia virus infection with a limited role of ifn-c and nitric oxide synthetase elevated generation of reactive oxygen/nitrogen species in hantavirus cardiopulmonary syndrome wild-type puumala hantavirus infection induces cytokines, c-reactive protein, creatinine, and nitric oxide in cynomolgus macaques nitric oxide and macrophage function the role of nitric oxide in innate immunity inhibition of vesicular stomatitis virus infection by nitric oxide effects of human and murine interferons against hemorrhagic fever with renal syndrome (hfrs) virus (hantaan virus) an antiviral mechanism of nitric oxide: inhibition of a viral protease viral mutation accelerated by nitric oxide production during infection in vivo s-nitrosylation of viral proteins: molecular bases for antiviral effect of nitric oxide induction and regulation of nitric oxide synthase in airway epithelial cells by respiratory syncytial virus respiratory syncytial virus infection of human respiratory epithelial cells enhances inducible nitric oxide synthase gene expression nitric oxide synthesis enhances human immunodeficiency virus replication in primary human macrophages hepatitis c virus infection activates the immunological (type ii) isoform of nitric oxide synthase and thereby enhances dna damage and mutations of cellular genes isolation of the etiological agent of korean hemorrhagic fever characterization of dobrava virus: a hantavirus from slovenia, yugoslavia isolation and characterization of dobrava hantavirus carried by the striped field mouse (apodemus agrarius) in estonia cell culture adaption of puumala hantavirus changes the infectivity for its natural reservoir, clethrionomys glareolus, and leads to accumulation of mutants with altered genomic rna s segment vaccination of c /bl mice with dobrava hantavirus nucleocapsid protein in freund's adjuvant induced partial protection against challenge biochemistry of nitric oxide and its redox-activated forms antigenic variation of european haemorrhagic fever with renal syndrome virus strains characterized using bank vole monoclonal antibodies delayed viremia and antibody responses in puumala hantavirus challenged passively immunized cynomolgus macaques key: cord- - qbbgp authors: shibata, isao; tsuda, tomoyuki; mori, masahumi; ono, masaaki; sueyoshi, masuo; uruno, katsuyoshi title: isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: qbbgp this paper describes the isolation of porcine epidemic diarrhea (ped) virus in vero and porcine cell cultures, and the influence of age on disease in experimental infection. ped virus was isolated from the small intestine of piglets inoculated with ped samples and cultured in vero, porcine bladder and kidney cells propagated in collagen-coated tissue culture plates in maintenance medium (mm) containing trypsin. in porcine bladder and kidney cell cultures inoculated with isolated ped virus, cytopathic effects (cpe) including cell fusion were detected. specific brilliant fluorescence was observed in the cytoplasm of these cells. two- and -day old, and -, -, - and -week old specific pathogen-free (spf) pigs were orally inoculated with ped virus isolated from an outbreak. all - and -day old pigs inoculated developed severe watery diarrhea from post-inoculation day (pid) and died between pid and . although three of five -week old pigs developed diarrhea on pid – , they eventually recovered. in the -week old group, three of five pigs had mild diarrhea for – days. none of the - and -week old pigs showed any clinical signs. antibodies against ped virus were detected in all surviving pigs by virus neutralization (vn) test and immunofluorescence assay (ifa). therefore, there is an age-dependent resistance to pathogenic ped virus infection in pigs. isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages porcine epidemic diarrhea (ped) virus is a member of the coronaviridae, and is antigenically distinguishable from the other porcine coronaviruses, transmissible gastroenteritis (tge) virus and hemagglutinating encephalomyelitis virus cavanagh, ) . experimental infections with ped virus isolates have indicated that the virus is a primary etiologic agent of diarrhea in pigs (debouck and pensaert, ; debouck et al., ; coussement et al., ) . the principal features of ped are watery diarrhea, dehydration and high mortality in suckling pigs. in , a coronavirus-like particle was ®rst identi®ed during episodes of epizootic diarrhea in pigs in belgium (pensaert and debouck, ) and the uk (wood, ; chasey and cartwright, ) . ped has subsequently been reported in canada (turgeon et al., ) , hungary (horvath and mocsari, ) , germany (pospischil et al., ) and korea (kweon et al., ) . in japan, there was an outbreak of ped-like disease from late to early (takahashi et al., ; kuwahara et al., ) . further outbreaks occurred between late and (sueyoshi et al., ; tsuda, ) . in an acute outbreak of ped in , more than the , suckling pigs died (tsuda, ) . attempts at propagating ped virus in porcine cell or organ cultures have been unsuccessful. the ®rst adaptation of ped virus to vero cell cultures using medium containing trypsin was reported in (hofmann and wyler, ) . in japan, ped virus was ®rst isolated in vero cell culture in the same manner (kusanagi et al., ) . this paper describes the isolation of ped virus not only in vero cells but also in cultures derived from pig bladder and kidney cells by addition of trypsin to the medium. furthermore, to determine the effect of pig age on disease, different age groups of pigs were inoculated with the japanese isolate of ped virus. growth medium (gm) was eagle's minimum essential medium (mem) containing . % tryptose phosphate broth, % inactivated fetal calf serum (fcs) and antibiotics was used for the propagation of cells. maintenance medium (mm) consisted of eagle's mem with . % tryptose phosphate broth, . % yeast extract, and mg/ml trypsin (difco, usa), unless otherwise stated. established cell lines of vero cells derived from african green monkey kidney (rcv ) and ma from fetal macacus rhesus monkey kidney were used. sb and sb cells prepared from the bladder epithelial cells of cesarean-derived colostrumdeprived (cdcd) piglets were used for virus isolation at approximately ± passages. sk cells prepared from the cdcd pig kidney were used at approximately ± passages. the small intestines obtained from four piglets with naturally acquired infection showing severe diarrhea were homogenized with mm. ped virus antigens were detected in the enterocytes of these pigs by immunohistochemistry. the % pooled homogenates were centrifuged and the resulting supernatants were passed through a nm membrane ®lter. six -day old cdcd piglets were orally inoculated with ml of the homogenized samples and sacri®ced on post-inoculation days (pid) ± . the small intestines collected from these infected pigs were treated as described above and the pooled homogenates were used as virus stock. the virus stock was stored at À c until experimental inoculation of pigs. the virus stock was approximately . ld / ml for a -day old pig. for virus isolation, three -day old cdcd piglets were inoculated with the virus stock and sacri®ced on pid (second passage in pig). homogenized samples were prepared for virus isolation according to the method described previously (hofmann and wyler, ). after removal of gm, con¯uent cell cultures in collagen-coated -well tissue culture plates (iwaki glass, japan) were washed once with mem. then, the cells were inoculated with . ml per well of the clari®ed homogenate. after adsorption at c for h, the inoculum was removed and the monolayers were washed three times with ml of mem. the cell cultures were fed with ml per well of mm. control cultures were mock-inoculated with the same volume of mm instead of viral inoculum. if no cytopathic effect (cpe) was detected within days, ®ve blind passages were performed using supernatant¯uids of cell culture in the same manner as the original samples. twenty eight speci®c pathogen-free (spf) pigs in different age groups (six at days old, two at days old, ®ve each at , , and weeks old) were obtained from an spf pig herd. each group of pigs was housed separately in a containment room and fed a commercial milk or solid diet. the ambient room temperature was maintained at ± c for -and -day old pigs and ± c for the other pigs. two-day old pigs were divided into uninoculated control or infection groups. two-day old and all other pigs were orally inoculated with ml of -and -fold diluted virus stock, respectively. blood samples were collected weekly from each pig. clinical signs were recorded twice a day until slaughter. surviving pigs were euthanized and necropsied at post-inoculation week (piw) or . vero cell cultures on coverslips were ®xed in acetone for min, about h after virus inoculation. they were then stained with the experimentally inoculated pig serum at c for min. after incubation, they were washed with phosphate buffered saline (pbs) three times and stained with protein a conjugated with¯uorescein isothiocyanate (zymed, usa) at c for min. after washing, the cells were mounted with buffered glycerol and examined under a¯uorescence microscope. the vn test was carried out by the microtiter method using vero cells. sera were heated at c for min before use. serial two-fold dilutions of serum were mixed with an equal volume of z p strain virus suspension containing tcid / . ml. the mixture were incubated at c for h and . ml of virus±serum mixture was inoculated into each of the two wells. following adsorption at c for h, the inocula were removed and the monolayers were washed three times with mem. then, . ml of mm containing mg/ml trypsin was added to each well and the cultures were incubated at c for days. the antibody titer was expressed as the reciprocal of the highest serum dilution inhibiting cpe in at least one of the two wells. the avidin±biotin (ab) technique was used for the detection of ped virus antigen in tissues. an abc kit (vector laboratories, usa) was used to examine formalin-®xed, paraf®n wax-embedded sections of the gastrointestinal tract. sections were counterstained with methyl green. rabbit antiserum against ped virus (tsuda, ) was used as primary antibody. histopathological examination was performed according to routine procedures. in brief, tissue samples were ®xed in % neutral phosphate-buffered formalin. thin sections of paraf®n-embedded samples were stained by hematoxylin and eosin. as shown in table , cytopathic agents were isolated in vero, sk and sb cell cultures inoculated with second passage intestinal samples. at ®rst passage, cpe was inapparent vero À a sk À À sb À sb À À À ma À À À because of the cytotoxic effect of the inoculum. on passage in vero cell culture, cpe characterized by cell fusion and syncytial formation was detected microscopically after an incubation period of ± days. cpe progressed more slowly in sk and sb cell cultures than in vero cell culture. the number of cells which were rounded, detached and fused were increased after ± days of incubation (fig. ) . although syncytial formation was not apparent under microscope in non-stained cultured cells, fused cells were obvious in sb cell culture samples stained with wright giemsa's solution (fig. ) . vero cells inoculated with viruses isolated in sb or sk cells showed the same cpe with syncytial formation. by ifa, speci®c brilliant¯uorescence were observed in the cytoplasm of these cells (fig. ) and the isolates were identi®ed as ped virus. nō uorescing cells were observed in uninoculated control cells. the isolated ped virus from pig no. in vero cell culture was clone-puri®ed three times by plaque selection and designated z p . cpe was not detected microscopically in sb and ma cells at the ®fth passage. all -and -day old pigs inoculated with ped virus developed severe watery diarrhea from pid and died between pid and (table ). ped virus antigen was detected by the ab technique in the small intestine of dead pigs. vomiting was occasionally seen on pid ± . non-inoculated control -day old pigs kept under the same conditions remained healthy. three of ®ve -week old pigs developed diarrhea on pid ± which lasted ± days. although all these pigs were slightly depressed and anorectic after inoculation, they recovered after week. ped virus antigen was detected by the ab technique in the jejunum and ileum of pig no. which was euthanized on pid . in the -week old group, three of ®ve pigs had mild diarrhea which started on pid ± and continued for ± days. in this group, other clinical signs such as depression and anorexia were not observed. all of -and -week old pigs showed no clinical signs including diarrhea throughout the experiment. all the pigs in the -, -, -and -week old groups survived. before infection, all of the pigs were negative for antibody against ped virus by both vn test and ifa. the antibodies were ®rst detected on piw or and they peaked between piw and on vn test and on piw on ifa (fig. ) . ifa titers were higher than those of vn antibody. all of the non-inoculated control pigs were negative for both antibodies throughout the study. in the present study, ped virus was successfully propagated not only in vero cells but also in sb and sk cell cultures. hofmann and wyler ( ) ®rst isolated and adapted ped virus to serial propagation in vero cells, which are derived from african green monkeys, by adding trypsin to the mm. however, attempts at isolating ped virus in porcine cell cultures in the presence or absence of trypsin have been unsuccessful until now. previous reports suggested that porcine cell cultures were damaged by trypsin, which then might not be able to support virus replication (hofmann and wyler, ) . in the present study, collagen-coated plates were used; cells cultured on collagen-coated plates are more resistant to trypsin (data not shown). this might be the main reason for the successful isolation of ped virus in porcine cell cultures in the present study. this method may be suitable for cell culture in medium containing trypsin or other proteinases. ped virus was isolated in sb cells but not in sb cells. although ped virus propagated in sb cells showed cpe in sb cells, the titer in these cells was lower than in sb cells (data not shown); the cells had been derived from the bladder of different pigs and were used at the same passage numbers. the reason for the difference in susceptibility for viral propagation between these cells is not known. upon experimental infection, -and -day old pigs inoculated with ped virus developed severe diarrhea and died. the -and -week old pigs showed mild diarrhea after inoculation and survived. the -and -week old pigs did not show any clinical signs, but developed antibody against ped virus. coussement et al. ( ) described that -or -day old cesarian-derived piglets infected oronasally with the cv strain of ped virus showed severe diarrhea after an incubation period of ± h. debouck and pensaert ( ) reported that pigs between and days old experimentally inoculated with ped virus developed diarrhea and some pigs younger than days old died. these results are similar to those of the present study. in several outbreaks, ped has caused diarrhea in pigs of all ages (pensaert and debouck, ; takahashi et al., ) . in the present study, however, severe diarrhea was only observed in -and -day old pigs. the difference in the clinical signs between ®eld cases and the present examination may re¯ect differences in the infectious dose, the susceptibility of pigs, the environmental conditions or the virulence of the strains. the majority of herd outbreaks of ped occur during the colder months, especially between january and april (tsuda, ) . in tge virus infection, (shimizu et al., ; shimizu and shimizu, ) showed that a high ambient temperature resulted in increased disease resistance, while a low ambient temperature or temperature changes caused a dramatic enhancement in clinical signs. in the present study, ± -week old pigs were kept at ± c throughout the study. consequently, a stable comfortable ambient temperature might induce resistance to clinical disease after ped virus infection. classi®cation and nomenclature of virus. fifth report of the international committee on taxonomy of viruses virus-like particles associated with porcine epidemic diarrhea pathology of experimental cv coronavirus enteritis in piglets experimental infection of pigs with a new porcine enteric coronavirus cv the pathogenesis of an enteric infection in pigs, experimentally induced by the coronavirus-like agent, cv propagation of the virus of porcine epidemic diarrhea in cell culture ultrastructural changes in the small intestinal epithelium of suckling pigs affected with a transmissible gastroenteritis (tge)-like disease isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate passage in piglets of a coronavirus associated with porcine epidemic diarrhea isolation of porcine epidemic diarrhea virus (pedv) in korea a new coronavirus-like particle associated with diarrhea in swine an immunoelectron microscopic and immuno¯uorescent study on the antigenic relationship between the coronavirus-like agent, cv , and several coronaviruses light microscopy and ultrahistology of intestinal changes in pigs infected with epizootic diarrhoea virus (evd): comparison with transmissible gastroenteritis (tge) virus and porcine rotavirus infection effects of ambient temperatures on clinical and immune responses of pigs infected with transmissible gastroenteritis virus effects of ambient temperatures on induction of transmissible gastroenteritis in feeder pigs an immunohistochemical investigation of porcine epidemic diarrhoea an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan porcine epidemic diarrhea: its diagnosis and control coronavirus-like particles associated with diarrhea in baby pigs in quebec an apparently new syndrome of porcine epidemic diarrhoea key: cord- -ot pvtbl authors: chen, f; chan, k. h; jiang, y; kao, r.y.t; lu, h. t; fan, k. w; cheng, v.c.c; tsui, w.h.w; hung, i.f.n; lee, t.s.w; guan, y; peiris, j.s.m; yuen, k. y title: in vitro susceptibility of clinical isolates of sars coronavirus to selected antiviral compounds date: - - journal: journal of clinical virology doi: . /j.jcv. . . sha: doc_id: cord_uid: ot pvtbl abstract effective antiviral agents are urgently needed to combat the possible return of severe acute respiratory syndrome (sars). commercial antiviral agents and pure chemical compounds extracted from traditional chinese medicinal herbs were screened against clinical isolates of sars coronavirus by neutralisation tests with confirmation by plaque reduction assays. interferon-beta- a, leukocytic interferon-alpha, ribavirin, lopinavir, rimantadine, baicalin and glycyrrhizin showed antiviral activity. the two interferons were only active if the cell lines were pre-incubated with the drugs h before viral inoculation. results were confirmed by plaque reduction assays. antiviral activity varied with the use of different cell lines. checkerboard assays for synergy were performed showing combinations of interferon beta- a or leukocytic interferon-alpha with ribavirin are synergistic. since the clinical and toxicity profiles of these agents are well known, they should be considered either singly or in combination for prophylaxis or treatment of sars in randomised placebo controlled trials in future epidemics. although the sars epidemic has been successfully contained with quarantine and infection control measures, the presence of this virus in wild game food animals , stocks in laboratories and possible seasonality of this disease suggest that recurrence of such an epidemic is not unlikely in the coming winters. since all age groups are affected and a high fatality is noted in the elderly and those with co-morbidities (donnelly et al., ) , there is an urgent need to find a cure. prospective clinical and viral load studies in nasopharyngeal secretions from sars patients showed that viral replication peaked at the th day after the onset of symptoms with subsequent clinical deterioration in % of the cases despite a decreasing viral load (peiris et al., ) . therefore the key facet of management should include respiratory support, immuno-modulation in selected cases and early institution of an effective antiviral agent. such an antiviral agent, if given early, may decrease the peak viral load and the associated immuno-dysregulatory damage. at the moment, there are no commercially available antiviral agents tailored-made for sars coronavirus. there is an urgent need to search for an agent with a known in use clinical and toxicity profile so that a randomised placebo control trial can be conducted if the epidemic recurs in one of the coming winters. we report in this study on the in vitro antiviral susceptibility of isolates of sars coronavirus to commercially available antiviral agents and pure chemical compounds including baicalin, glycyrrhizin, and chlorogenic acid extracted from traditional chinese herbs. ten isolates of sars coronavirus from different sars patients who satisfied the revised who criteria for sars are listed in table . the drugs used for antiviral susceptibility specialists to draw up a "technical scheme (tentative) for the prevention and treatment of severe acute respiratory syndrome (sars) using traditional chinese medicine". in the scheme, a recipe "qing fei jie du tang" (soup for clearing the lung and detoxification) was recommended which consists of huang qi (astragalus membranaceus) gm, chai hu (bupleurum chinense) gm, ma huang (ephedra sinica) gm, xing ren (prunus armeniaca) gm, sheng she gao (plaster stone) gm, sheng yi ren (coix lacryma-jobi) gm, gua wei pi (benincasa hispida) gm, jie geng (platycodon grandiflorum) gm, bo he (mentha haplocalyx) gm, huang qin (scutellaria baicalensis) gm, sheng gan cao (glycyrrhiza uralensis) gm, jin yin hua (flos lonicerae) gm, and qing hao (artemisia apiacea) gm. among them, only scutellaria baicalensis, glycyrrhiza uralensis, flos lonicerae and artemisia apiacea have their pure chemically defined ingredients being extracted, purified and documented to have antimicrobial activities. consequently, the main bioactive compounds, namely, baicalin (derived from scutellaria baicalensis), glycyrrhizin (from glycyrrhiza uralensis), chlorogenic acid (from flos lonicerae) and artesunate (from artemisia apiacea) were investigated in the present study. the pharmacological properties of baicalin, glycyrrhizin, and chlorogenic acid are summarized in table . artesunate is not included in this table since it is already well known as an anti-malarial drug in western medicine (price, ) . they were extracted as we have previously reported (lu et al., ) . the concentrations of baicalin, glycyrrhizin, no glycyrrhizin in plasma is found after oral administration of mg glycyrrhizin in healthy persons, presumably glycyrrhizin is metabolized to glycyrrhetinic acid by intestinal bacteria which contain ␤-d-glucuronidase or the amount consumed is too little only traces expected ( mg per person, in human); this may be due also to that the amount consumed is much lower than that for animals standard doses in oral administration in humans ∼ mg baicalin (as tablets); also can be up to ∼ mg baicalin (calculated from herb, assuming g herb used; the herb may contain up to % as baicalin) ∼ mg glycyrrhizin (as tablets) or ∼ mg glycyrrhizin (calculated from the herb assuming that the herb contains . % glycyrrhizin) ∼ mg (calculated from the herb assuming that the herb contains . % chlorogenic acid) serum level (after intravenous administration) chlorogenic acid, and lopinavir in the cell culture system were also monitored by hplc (lu et al., ) whereas the concentration of others were monitored by neutralization assays with the vesicular stomatitis virus indiana strain and a laboratory strain of influenza a h n .the procedures used for in vitro antiviral susceptibility testing are as follows. initial screening of all compounds against the prototype sars coronavirus strain no. was performed in -well microtitre plates seeded with foetal rhesus kidney- cells. two-fold dilutions of antiviral agents starting from more than four times the peak serum concentration after the maximum therapeutic dose to less than one-quarter of the trough serum concentration were tested in quadruplicate against tcid of sars coronavirus. a corresponding set of cell controls with drug but without virus inoculation was used as controls for drug toxicity. the cells were scored for the inhibition of the cytopathic effect (cpe) at and h. those compounds with demonstrable in vitro inhibitory activity were re-assayed against the other nine strains of sars coronavirus collected from different patients from different hospitals of the hong kong special administrative region (hksar). their antiviral activities were also compared in both foetal rhesus kidney- (frhk- ) and vero-e cell lines. those likely to have clinically significant inhibitory activity were tested by the plaque reduction assay. for selected agents with consistent activity in the plaque reduction assay, checkerboard assays for synergy were per-formed for combinations of interferons and ribavirin using the same neutralization test in well microtiter plates seeded with vero cell line. cells were not incubated with the interferons before viral inoculation. vero cells were used instead of vero e and frhk- cell lines because better antiviral effect can be demonstrated in vero but less so in the other two cell lines for ribavirin and the interferons. ten isolates of sars coronavirus from different patients with sars admitted to different hospitals in hk-sar showing seroconversion towards the prototype virus infected frhk- cell line were used in this study (table ) . they were isolated from the lung tissue biopsy (prototype virus, m ), urine, and nasopharyngeal aspirates. initial screening of commercially available antimicrobial agents against the prototype virus grown in frhk- cell line did not reveal inhibitory activities for acyclovir, ganciclovir, cidofovir, foscarnet, interferon-alpha- a, interferon-alpha- b, amantadine, zidovudine, stavudine, nevirapine, abacavir, and ritonavir. inhibitory activities were not detectable for glycyrrhizin, artesunate and chlorogenic acid in frhk- cell line. glycyrrhizin was still included for further testing because this was reported to be active in vero-e cell lines (cinatl et al., a) . further testing by neutralization tests table comparison of antiviral activity of compounds against strains of sars-cov in frhk cell line, against the prototype strains ( ) with the other isolates of sars coronavirus against the active compounds confirmed detectable inhibitory activities for leukocytic interferon-alpha, interferon-beta- a, ribavirin, lopinavir, rimantadine, and baicalin. the range of their effective concentration of compound required to reduce the plaque forming unit by % (ec ) at and h, and their selectivity index are shown in table . when the same neutralization test on these compounds was run in vero-e cell line, rimandatine, glycyrrhizin, leukocytic interferon-alpha and interferon-beta were more active especially at h. moreover, pre-incubation of the cell lines with these two interferons for h before adding the virus markedly enhanced the inhibitory activity by three to over -fold. but ribavirin, lopinavir, and baicalin were less active in the vero-e cell line (table ) . as for the plaque reduction assay, vero cell lines were used instead of vero e or frhk- cell lines because antiviral activity can be demonstrated for most of the agents. only interferon-beta- a, leukocytic interferon-alpha, lopinavir, ribavirin, rimantadine, and baicalin were tested. the ec of interferon-beta- a ( u/ml), leukocytic interferon-alpha ( u/ml), lopinavir ( g/ml), ribavirin ( g/ml), rimantadine ( g/ml), and baicalin ( g/ml) are comparable to the results obtained from cpe assays. tests for synergism between ribavirin and lopinavir have already been reported (chu et al., ) . no synergism could be demonstrated between rimantadine and ribavirin. the most active compounds are the interferons. thus further checkerboard assays were performed with combinations of leukocytic interferon-alpha or interferon-beta- a, and ribavirin. marked synergism was seen at both and h. the combination of leukocytic interferon-alpha ( g/ml) and ribavirin ( g/ml) or interferon-beta- a ( . g/ml) and ribavirin ( g/ml) were shown to be active at h of incubation (table ) . control of sars may be achieved by epidemiological measures, antiviral prophylaxis or treatment, and vaccination. during the last pandemic of sars, the only available means for control were public health measures such as isolation of suspected cases, quarantine of contacts, and personal protective infection control procedures for high-risk individuals such as health care workers. there is an urgent need to find effective antiviral agents with acceptable side effect profiles. in developing countries such as china, commercially available western antiviral medicine is unlikely to be affordable by most people. moreover, the sars mortality of mainland china was only % comparing favourably with the % to % of other areas (who, ) . china is also the only place where traditional chinese medicinal herbs were extensively used for treatment of sars. the development of vaccine will take a much longer time. therefore, we undertook these antiviral susceptibility tests for all commercially available antiviral agents in the hksar and pure chemicals purified from traditional chinese herbs known to have antimicrobial activity. these chosen herbs were included in a standard formula used for the treatment of sars in china and the hksar. only interferon-beta and glycyrrhizin were reported to have significant antiviral activity against sars coronavirus (cinatl et al., a,b) . using the frhk- cell line, we have shown that ribavirin, rimantadine, lopinavir, and baicalin also have detectable antiviral activities. however, like the interferons and glycyrrhizin, their activities tend to decrease with incubation beyond h (table ) . judging from the achievable serum levels with standard oral or parenteral dosing, rimantadine, ribavirin, glycyrrhizin, and even the two interferons are unlikely to have clinically significant in vivo activities. moreover, lopinavir, and rimantadine have a relatively inferior selectivity index of to . upon subsequent testing with vero-e cell line, both leukocytic interferon-alpha and interferon-beta- a were more active and especially after pre-incubation for h before viral inoculation. the findings suggest that prophylaxis with the interferons should be considered. though ribavirin was much less active in the vero cell line, it is highly synergistic with either two interferons. therefore, a combination of ribavirin with either of these two interferons should be considered for the treatment of sars. interferon-gamma was reported not to possess antiviral activity against sars coronavirus (cinatl et al., b) , whereas interferon-beta was confirmed to be active in this study. what is interesting was the demonstration of activity of leukocytic interferon-alpha despite the lack of activity of the recombinant interferon-alpha- a and interferon-alpha- b. this was not unexpected because this preparation of leukocytic interferon-alpha is a multi-subtype natural interferon with predominantly interferon alpha- and alpha- in contrast to the other commercial preparation with a single subtype of recombinant interferon-alpha- . in in vitro studies, different subtypes have been found to have different antiviral activities as well as immunological effects (foster et al., ) . it was also demonstrated that leukocytic interferon-alpha had a superior antiviral effect than that of recombinant interferon on human immunodeficiency virus infection (fan et al., ) . it is important to know that in vitro findings may not correspond with clinical efficacy. despite its in vitro activity, topical or systemic interferon-alpha did not produce a consistent reduction in symptoms or lesion duration of genital herpes (eron et al., ; lebwohl et al., ) . and interferon-alpha was not effective in preventing cmv infections or treating cmv pneumonia in bone marrow transplant patients (meyers et al., ) . despite its broad-spectrum antiviral activities against respiratory viruses in vitro, prophylactic intranasal interferon-alpha is only protective against rhinovirus-induced common cold under natural condition (douglas et al., ) . this was unexpected because intranasal leucocyte or recombinant interferon-alpha protect against experimental human infection by rhinovirus, coronavirus, and respiratory syncytial virus (hayden and gwaltney, ; higgins et al., ; higgins et al., ) . besides the high cost of interferons, the high incidence of fever and flu syndrome of up to % during its initial phase of administration may pose confusions in terms of the response to treatment. the well-known side effect of pancytopenia may also be confused with markers of sars activity such as a decrease in platelets and occasionally neutrophils (raanani and ben-bassat, ) . although interstitial pneumonitis and bronchiolitis obliterans organising pneumonia are rare complications of prolonged use of interferons (karim et al., ; ogata et al., ) , there is always a fear that their proinflammatory effect may worsen the viral pneumonitis caused by sars. as for the less expensive option, baicalin but not glycyrrhizin may be considered if traditional chinese medicine is to be used for antiviral prophylaxis or treatment. the serum level after mg of glycyrrhizin orally was not detectable. even with a mg dose of intravenous administration, the peak serum level is only g/ml which is still below the ec of glycyrrhizin. although an oral dose of . gm of baicalin can only achieve a serum concentration of . g/ml, intravenous administration of a mg dose of baicalin in human can achieve a peak serum concentration of g/ml. thus intravenous baicalin should be con-sidered for treatment in randomised placebo control trials in developing countries where such formulations are available and affordable. baicalin was shown to inhibit hiv- by two mechanisms (kitamura et al., ; li et al., ) . at the level of cellular entry, baicalin can conjugate with selected chemokines such as mip- ␤ and sdf- ␣, and interfere with their capacity to activate cellular receptors ccr and cxcr respectively. these two co-recpetors are essential elements for hiv- infection and therefore baicalin can inhibit env-protein mediated fusion of hiv with cells expressing cd /ccr or cd /cxcr . baicalin has also been known to inhibit hiv- reverse transcriptase probably by interfering with the binding of viral rna to the rt molecule near the active site of the enzyme. in terms of prophylaxis against sars short of an effective vaccine, intranasal leukocytic interferon-alpha or interferon-beta- a are likely to be effective. however the local side effect of nasal irritation can decrease compliance. it could also be considered for randomised placebo-control trials. as for the antiviral treatment of symptomatic sars, it is important to have a rapid and reliable diagnostic test since early institution of antiviral therapy is important to decrease the peak viral load . interferon-beta- a or leukocytic interferon-alpha plus ribavirin appear to be the most effective combination. since interferons may not be effective in inducing an antiviral state in the uninfected host cells during the first h, a combination with a short course of ribavirin appears to be reasonable. this will also reduce the side effects and fluid volume associated with a full course of ribavirin. despite the superiority of interferons in the in vitro assays, there is little clinical data of its use in the treatment of acute viral respiratory infection in human. thus the combination of ribavirin with lopinavir/ritonavir should still be considered since some positive clinical data has already been accumulated in a historical controlled treatment trial (chu et al., ) . glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus treatment of sars with human interferons the role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong prophylactic efficacy of intranasal alpha -interferon against rhinovirus infections in the family setting therapy of genital herpes with topically applied interferon increased efficacy of human natural interferon alpha (ifn-alpha n ) versus human recombinant ifn-alpha for inhibition of hiv- replication in primary human monocytes different relative activities of human cell-derived interferon-alpha subtypes: ifn-alpha has very high antiviral potency isolation and characterization of viruses related to the sars coronavirus from animals in southern china intranasal interferon-alpha treatment of experimental rhinoviral colds intranasal interferon as protection against experimental respiratory coronavirus infection in volunteers the efficacy of intranasal interferon alpha- a in respiratory syncytial virus infection in volunteers interstitial pneumonitis in a patient treated with alpha-interferon and ribavirin for hepatitis c infection baicalin, an inhibitor of hiv- production in vitro recombinant alpha- interferon gel treatment of recurrent herpes genitalis flavonoid baicalin inhibits hiv- infection at the level of viral entry application of high-speed counter-current chromatography to the preparative separation and purification of baicalin from the chinese medicinal plant scutellaria baicalensis toxicity and efficacy of human leukocyte interferon for treatment of cytomegalovirus pneumonia after marrow transplantation interferon-related bronchiolitis obliterans organizing pneumonia clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study early diagnosis of sars coronavirus infection by real time rt-pcr artemisinin drugs: novel antimalarial agents immune-mediated complications during interferon therapy in hematological patients world health organization. summary table of sars cases by country we acknowledge research funding from the kai cheong tong sars research fund, the university of hong kong. key: cord- -cdn epy authors: artuso, maría c.; ellenberg, paula c.; scolaro, luis a.; damonte, elsa b.; garcía, cybele c. title: inhibition of junín virus replication by small interfering rnas date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: cdn epy junín virus (junv), the etiological agent of the argentine hemorrhagic fever, has a single-stranded rna genome with ambisense expression which encodes for five proteins. in previous works we have demonstrated that the z arenavirus matrix protein represents an attractive target for antiviral therapy. with the aim of studying a new alternative therapeutic mechanism, four z-specific sirnas (z - to z -sirnas) were tested showing variable efficacy. the most effective inhibitor was z -sirna targeted at the region encompassed by nt – of z gene. the efficacy of this z -sirna against junv was also demonstrated in virus-infected cells, by testing infectious virus plaque formation ( . % junv yield reduction), viral rna level or antigen expression, as well as in cells transfected with z-specific reporter plasmids ( % reduction in expression of z-egfp fusion protein). furthermore, the lack of effect of this z-sirna on the expression of other junv proteins, such as n and gpc, confirmed the specificity of action exerted by z -sirna on z transcript. thus, the present study represents the first report of virus inhibition mediated by rna interference for a new world arenavirus. viruses are among the most important causes of morbidity and mortality worldwide, but especially in the developing countries where the national health care system is deficient. particularly, arenaviruses are among the etiological agents that can be considered within the neglected viral infections in south american countries where the human hemorrhagic fevers are often fatal to the infected patients. junín virus (junv) is the agent of argentine hemorrhagic fever (ahf), with mortality rates ranging from to % in the absence of the administration of standardized doses of convalescent plasma that is today the best therapy against ahf (damonte and coto, ) . junv is an enveloped, single stranded, ambisense rna virus with a segmented genome consisting of two segments, designated large (l) and small (s). the s segment encodes the nucleocapsid protein (n) and a pre-envelope glycoprotein precursor (gpc) which is processed post-translationally into a signal peptide, the external glycoprotein g and the transmembrane g , whereas the l fragment encodes the rna polymerase l and the matrix protein called z. the z protein has been implicated in several aspects of arenavirus biology. it has been shown that z exerted an inhibitory effect on viral rna synthesis de la torre, , ; lópez et al., ) . in addition to this regulatory role, z was also implicated as a virion component with matrix functions, similar to other enveloped negative-strand viruses (neuman et al., ; perez et al., ; salvato et al., ; salvato, ; strecker et al., ) . consistent with its features, z presents a ring finger and a canonical late domain motif that enable z to interact with the host cell (borden et al., ; campbell-dwyer et al., ; djavani et al., ) and viral proteins. furthermore, z-mediated budding requires its myristoyl modification (perez et al., ) , which likely facilitates z association with membranes at budding sites. thus, z has an essential role in the particle formation. rna interference (rnai) is a post-transcriptional gene silencing process in which double-stranded rna (dsrna) initiates specific cleavage of cytoplasmic mrna. initially, dsrna is recognized and processed by dicer, an rnase iii enzyme (hutvágner et al., ) . the resulting processed dsrna consists of - nt fragments with - nt overhangs at the end of each rna strand (bernstein et al., ; zamore et al., ) . dicer-processed dsrna is recognized by the dsrna-induced silencing complex (risc) and used as a template to guide degradation of mrna that is homologous in sequence to the risc-bound dsrna fragment, resulting in greatly reduced protein production (hammond et al., ) . synthetically produced small interfering dsrna molecules (sir-nas) have been shown to induce the rnai effect in vitro and greatly decrease specifically targeted transcripts (elbashir et al., ) . such efforts have been expanded to include targeting of viral transcripts present in the cytoplasm for degradation by the risc complex. the list of the human viral pathogens that have (leung and whittaker, ) , poliovirus (gitlin et al., ) , adenovirus (chung et al., ) , human immunodeficiency virus (hannon and rossi, ; naito et al., ) , hepatitis b virus (li et al., ) , hepatitis c virus (liu et al., ; lupberger et al., ) , influenza a (ma et al., ) , marburg virus (fowler et al., ) , human papillomavirus (jiang and milner, ) , and coronavirus (zhang et al., ) , among others. to the present, only two reports were published describing the use of sir-nas with the old world arenaviruses lymphocytic choriomeningitis virus (lcmv) (sánchez et al., ) and lassa virus (lasv) (müller and günther, ) . since the matrix z protein is involved in processes that are fundamental to the success of viral infection, we selected z as candidate target gene in order to determine the efficacy of rnai for inhibition of junv gene expression. here we report the identification of sirna target regions on z that are able to silence its expression and impair the production of infectious viral progeny. vero and bhk- cells were grown as monolayers in eagle's minimum essential medium (mem) (gibco) containing % fetal bovine serum (gibco) and g/ml gentamycin (sigma). medium was supplemented with m hepes buffer (sigma) when incubated at • c under % co . maintenance medium (mm) consisted of mem with . % fetal bovine serum. the attenuated strains iv and xjcl of junv (candurra et al., ) , the trlv strain of tacaribe virus (tcrv), and the armstrong b strain of lcmv were used. arenavirus stocks were prepared in bhk- cells and titrated by plaque formation in vero cells. four double-stranded sirnas were designed targeting the z transcript of junv-iv (genbank accession no. dq ) by using the algorithm programs from different companies. blast analysis was performed for ensuring the lack of homology with cellular genes. the sirnas targeted to z transcript (z -to z -sirnas) and a sirna without homology to any known gene transcript used as a non-silencing control (x-sirna) were supplied as annealed duplexes by invitrogen (table ) . vero cells grown in six-well plates at approximately % confluence were transfected with sirna using lipofectamine (invitrogen) according to manufacturer's instructions with minimum modifications. briefly, cell supernatant was removed and l opti-mem (gibco) containing l lipofectamine and m sirna was added. after h of incubation at • c, supernatant was removed and fresh advanced-mem (gibco) containing % fetal bovine serum was added to the cells. after h incubation at • c, transfected monolayers were infected with junv at a moi of . . at h post-infection (p.i.), supernatants were collected to determine virus yield by plaque assay and cells were processed to measure viral rna by real time pcr (qrt-pcr). vero cells grown as monolayers were infected with serial dilutions of the supernatants of z-sirna-transfected and junv-infected vero cells. after h adsorption, cells were washed, covered with mm containing . % methylcellulose, and incubated at • c under % co . at day p.i. virus plaques were counted and % inhibition with respect to the viral control was calculated. each value was the mean of two independent experiments performed in duplicate ± standard deviation. rna was isolated from sirna transfected and junv-infected vero cells using trizol (invitrogen) according to the manufacturer's instructions. to monitor rna replication, cdna was generated by using murine reverse transcriptase m-mlv ( u/l, invitrogen) and random primers. this cdna was amplified by real time pcr using sybrgreen (roche) detection. the mix reaction volume was l including l of cdna, dna polymerase gotaq ( u/l, promega) and specific primers to amplify gene z: z forward -atgggcaactgcaacggggcatc- and z reverse -agccaacagcaccaccaccatag- . real time pcr was carried out with an initial incubation at • c during min followed by cycles of s at • c, s at • c, s at • c, s at • c and a final incubation of s at • c. amplification plots were expressed as c t values to be analyzed with opticon monitor . . software where c t values represent the reaction cycle at which pcr products reach a threshold level of detection. c t values were normalized by using actin as standard. vero cell monolayers grown on coverslips were transfected with m z-sirna and infected with junv at h post-transfection as described in section . . at h p.i., cells were fixed with methanol for min at − • c for cytoplasmic immunofluorescence. indirect staining was carried out by incubation with a rabbit anti-junv polyclonal serum for h at • c followed by fluorescein (fitc)conjugated goat anti-rabbit igg in the same conditions. after a final washing, cells were mounted in a glycerol solution containing . % , -diazabicyclo[ , , ]octane (dabco) and visualized by a fluorescence microscope. a truncated z lacking a stop codon was obtained by pcramplification using plasmid pgem containing a full-length cdna copy of the z gene as template dna together with oligonucleotides z-f ( -ggatccatgggcaactgcaacggggcatc- ) and z-trunc-r ( -aaaagcggccgcctggtggtggtgctgttggct- ) containing a bamhi and noti site, respectively (underlined). the insert dna was subsequently subcloned into the bamhi and noti sites of the mammalian expression vector pcdna . to generate pcdna . -z trunc. oligonucleotides egfp-f ( -aaaagcggccgcatggtgagcaagggcgaggag- ) and egfp-r ( -aaaatctagattacttgtacagctcgtccatgcc- ), which contain a noti and xbai site, respectively (underlined), were used with plasmid pegfp c as template dna to pcr amplify the sequence encoding the enhanced green fluorescent protein (egfp). the amplicon was cloned into the noti-xbai digested pcdna . -z trunc vector to generate the reporter plasmid pcdna . -z-egfp, which resulted in the expression of a fusion protein z containing egfp in its cterminus. the same strategy was used to generate the reporter plasmid pcdna . -z-flag (dykddddk). in order to generate the n and gpc proteins encoding plasmids, the pcdnahismax a vector was modified and agei and clai restriction sites respectively (underlined) were added into the hindiii and apai vector sites using the following designated primers: -ctggctaactagagaacccactgc- and -tatgggccccccggcgcccccatcgatcccaccggtccccatggtttc-ggaggccg- . the n and gpc genes were obtained by pcramplification from junv-infected vero cells using the following primers: n agei sense ( -aaaaccggtatggcacactccaaagagg- ), n clai antisense ( -aaaatcgatcagtgcataggctgcc- ), gpc agei sense ( -gggaccggtatggggcaattcatcagc) and gpc clai antisense ( -tttatcgatgtgtcctcttgcgcc- ). then, pcr amplification fragments were cloned into modified pcdnahismax a vector to obtain the pcdnahismax a-n and pcdnahismax a-gpc constructs, respectively. vero monolayers grown to % confluence on mm diameter glass coverslips were co-transfected with g plasmid pcdna . -egfp, pcdna . -z-egfp, pcdna . -z-flag, pcdnahismax a-n or pcdnahismax a-gpc and m z-sirnas using lipofectamine (invitrogen) as described previously. as control, cells were transfected with the respective plasmid without z-sirna. to record egfp expression, at h post-transfection cells were fixed with methanol for min at − • c, mounted in glycerol with dabco and observed in a fluorescence microscope. for z-flag detection, cells fixed at h post-transfection were incubated with rabbit anti-flag polyclonal serum (cell signaling technology) overnight at • c, followed by incubation with rhodamine-conjugated goat anti-rabbit igg (sigma) for h at room temperature. then, cells were mounted and visualized. for n or gpc detection, cells fixed as above were incubated first with monoclonal antibodies sa bg or qc -bf (sánchez et al., ) , respectively, then with fitc-labeled goat anti-mouse igg. vero cells co-transfected with g pcdna . -z-flag with or without m z-sirna as described above were harvested at h post-transfection in sample buffer ( % sds, % -mercaptoetanol, % glycerol and . % bromophenol blue in . m tris-hcl, ph . ) (promega). after boiling the lysates during min, proteins were separated by % sds-page and blotted onto a pdvf membrane (millipore). membranes were incubated with a rabbit anti-flag serum overnight at • c. after washing with tris-buffered saline (tbs, mm tris-hcl, mm nacl, ph . ) containing . % tween , a second incubation was performed with horseradish peroxidase-conjugated anti-rabbit igg during h at room temperature. as control, the presence of actin was revealed by incubation with mouse monoclonal antiactin antibody (jla , calbiochem) followed by incubation with peroxidase-conjugated anti-mouse igg. for protein detection blots were treated with the western lightning luminescence system (perkinelmer). to evaluate the effect of z-sirnas on infectious junv production, vero cells were transfected with the respective sirna (z -sirna, z -sirna, z -sirna, z -sirna or x-sirna), and h later cultures were infected with junv at a moi of . . as a control, nontransfected junv-infected cells were also included. supernatants were collected at h p.i. and titrated by plaque assay. cells transfected with the non-specific x-sirnas did not exhibit a reduction in virus yield ( . × pfu/ml), showing similar levels to nontransfected junv-infected vero cells ( . × pfu/ml), indicating that transfection of the cells with the control x-sirna did not induce a non-specific interference with virus replication during the h incubation period. by contrast, reduction in virus titer was observed in cells treated with the sirnas corresponding to the z transcript (fig. a) . whereas z -sirna reduced the virus titer by . % compared to the viral control, z -sirna induced a . % yield reduction. in vero cells treated with both z -sirna and z -sirna, the virus yield was reduced by . %, showing that the combination of different silencing agents did not present any advantage (data not shown). on the other hand, z -sirna and z -sirna were less effective, with only . and . % of virus yield inhibition, respectively. interestingly, the silencing effect of z -sirna, the most effective inhibitor, was observed also when the virus yield inhibition was determined at longer period of incubation ( h p.i.) with . % of virus yield inhibition (fig. b) , and even when junv infection was performed at higher mois of and , with . and . % of virus yield inhibition, respectively (fig. c) . since the gene regions targeted for z-sirna design were conserved among arenaviruses (fig. ) , we looked for a possible interference of the selected z-sirnas on xjcl , another attenuated strain of junv, and also on the close antigenically related tcrv and the non-antigenically related lcmv. when z-sirnas originally designated against junv-iv were evaluated against junv-xjcl , a moderate virus yield inhibition was observed with z -to z -sirnas, with . , . , . and . % of virus yield inhibition, respectively. however, when junv z-sirnas were evaluated against the other two arenaviruses species, no inhibition was observed ( fig. a for tcrv and not shown for lcmv), confirming the specificity of the rna interference mechanism. since sirnas function by identifying and degrading mrna with its complementary sequence, we next examined the effect of z -sirna and z -sirna on the abundance of viral rna in junv-infected cells by qrt-pcr using z-specific primers. in contrast to vero cells transfected with the control non-silencing x-sirna, viral rna amplification was reduced in vero cells transfected with both zspecific sirnas (fig. d) . again, z -sirna was the most effective inhibitor with a -fold reduction in viral rna whereas z -sirna induced approximately a -fold rna inhibition with respect to control x-sirna (fig. d) . vero cells treated with z -sirna and infected with junv were also examined for viral antigen expression at h p.i. by an indirect immunofluorescence assay with a rabbit anti-junv polyclonal serum. hyperimmune junv antiserum allows the detection of the main viral proteins, the nucleoprotein n and the precursor and mature glycoproteins gpc and g . in accordance with the reduction in virus yield produced by z -sirna shown in fig. a , a concomitant the xjcl (dark grey bars) strains of junv, or tcrv (black bars). supernatants collected at h p.i. were titrated by plaque assay. inhibition of virus yield was calculated comparatively to the titer obtained in cells transfected with x-sirna. each value represents the mean of two independent experiments performed in duplicate ± standard deviation (s.d.). (b) vero cells were transfected with z -sirna and infected with junv, moi = . . supernatants were collected at or h p.i. and titrated by plaque assay. inhibition of virus yield was calculated as above. (c) vero cells were transfected with z -sirna and infected with junv at different mois. supernatants were collected at h p.i., and titrated by plaque assay. inhibition of virus yield was calculated as above. (d) amount of viral rna in vero cells transfected with z -sirna or z -sirna and infected with junv was quantified by real time rt-pcr. values, standardized to those of actin mrnas, were expressed as relative rna levels comparatively to the amount obtained in cells transfected with x-sirna and represented as mean of two independent experiments performed in triplicate ± s.d. (e) expression of viral antigens in vero cells transfected with z -sirna or x-sirna and infected with junv was detected by immunofluorescence assay using a rabbit anti-junv polyclonal serum. magnification = ×. decrease in cytoplasmic junv antigen expression was observed (fig. e) . to confirm that the z-sirnas were effective as specific inhibitors of the expression of z protein, the effect of z-sirnas on the expression of recombinant z fusion proteins was analyzed. first, the inhibitory action of z-sirna on the expression of the fusion protein z-egfp was determined by co-transfection of vero cells with the plasmid pcdna . -z-egfp and either the z-sirna or the non-silencing x-sirna. a series of co-transfections of the reporter plasmid pcdna . -egfp and the sirnas were assayed in parallel. at h post-transfection, egfp fluorescence was analyzed. the egfp protein was highly expressed in cells transfected with the reporter plasmid in the presence of co-transfected z-sirna or x-sirna (fig. a, panels a and b) . the control non-silencing x-sirnas had no apparent effect on z-egfp expression (fig. a, panel c) , whereas the four specific z-sirnas produced a marked reduction in fluorescence (fig. a , panels d-g). as observed in virus yield assay, z -sirna appeared to silence z-egfp gene expression more efficiently than the other z-sirnas. in fact, the quantification of fluorescent cells showed that z -to z -sirna reduced expression of the z-egfp fusion protein in comparison to x-sirna by . , . , . and . %, respectively (fig. b) . the specificity of z -sirna to block expression of z protein was further assessed by testing the expression of other recombinant protein, the z-flag fusion protein expressed by the plasmid pcdna . -z-flag, by immunofluorescence and western blot assays performed with rabbit anti-flag antibodies. it was evident from the results shown in fig. c and d that the z-specific sirna reduced the amount of detectable z-flag fusion protein in both assays whereas the co-transfection of vero cells with the control non-silencing x-sirna failed to block the synthesis of z-flag fusion protein. it is also worth to note that the expression in the same cell samples of a non-targeted host protein, actin (ca. kda), remained almost unaffected (fig. d) . to gain further evidence for the specificity of z-sirnas, we investigated whether the presence of z -sirna could decrease other transiently expressed junv proteins. to this end, cells were co-transfected with the z -sirna or x-sirna and plasmids pcd-nahismax a-n or pcdnahismax a-gpc encoding n and gpc junv proteins, respectively. as shown in fig. , no effect on n and gpc expression could be detected by indirect immunofluorescence using specific monoclonal antibodies against these proteins, respectively, suggesting that off-target effects are not the cause of the junv yield reduction observed in fig. . . effect of z-sirna on junv nucleocapsid and glycoprotein expression. vero cells grown on glass coverslips were untransfected (panels a and d), co-transfected with pcdnahismax c-gpc or pcdnahismax c-n and x-sirna (panels b and e) or z -sirna (panels c and f). at h post-transfection, cells were fixed, and protein expression was detected using specific monoclonal antibodies against gpc/g and n, followed by fitc-conjugated goat anti-mouse igg (magnification = ×). altogether, these data showed that the sirna targeting the z transcript exerts its inhibitory action exclusively through a significant and specific reduction in the expression of the z protein. in conclusion, results presented here have shown the effective inhibitory action against the arenavirus junv by sirnas directed against the sequence of the z transcript. four z-specific sirnas were tested showing variable efficacy. the most effective inhibitor was z -sirna, targeted to the region encompassed by nt - of z gene. the efficacy of this agent against junv was also demonstrated in virus-infected cells by testing infectious virus plaque formation, viral rna or antigen expression, as well as in cells transfected with z-specific reporter plasmids. furthermore, the lack of effect of this sirna on the expression of other junv proteins, such as n and gpc, confirmed the specificity of action exerted by z -sirna on z transcript. interestingly, the inhibitory effect observed in the release of junv infectious progeny by z -sirna (> % inhibition at h p.i.) is similar to the effect described in transient transfection of sirnas for other viruses, but higher than the activity recently reported for sirnas targeted to the termini of n and l genes in the arenavirus lasv (müller and günther, ) . it remains to be investigated if the antiviral potential of rna interference for junv infections here shown after transient transfection of synthetic sirna targeted to z gene can be improved by using a viral vector of a short hairpin rna that resembles sirnas precursors. however, sirnas elicit potent effects at relatively low doses; so as to minimize the interference with naturally occurring processes, current therapeutic efforts seem to be focused towards the use of a pool of synthetic unmodified naked sirnas (davis, ) . likewise, our results provide a clear evidence of the central role of z protein in the junv life cycle and assess the good perspectives of this protein as antiviral target in arenaviridae, in accordance with previous studies reporting the efficacy against junv and lcmv infections of compounds reactive with the ring finger motif of the z protein (garcía et al., (garcía et al., , . the present study represents the first report of virus inhibition mediated by rna interference for a new world arenavirus, a group in the family including four agents of severe viral hemorrhagic fevers in south america (junv, machupo, sabiá and guanarito viruses). it would be an ideal situation to get an antiviral approach effective against all these pathogens. regarding infectious diseases, ongoing clinical trials in the field of sirna are developed by different companies and most of them are in the preclinical stage, with the exception of hbv and hiv studies that are in phase i, and the most advanced program concerning the treatment of infection by rsv using sirna developed by alnylam pharmaceuticals that has just finished phase ii (lópez-fraga et al., ) . given the diversity of arenaviruses, it will be difficult to design a sirna treatment able to cross-inhibit several species in the family, even using a highly conserved sequence as antiviral target. in contrast with the cross-reactivity among the old world arenaviruses lasv, lcmv and mopeia virus reported with sirnas targeted to lasv (müller and günther, ) , in our study we observed no efficacy when junv designed z-sirnas were used to inhibit the close antigenically related new world arenavirus tcrv. even, the effectiveness against other junv strain was reduced when there is more than one nucleotide change in the target sequence, as shown for z -and z -sirna against iv and xjcl junv strains (figs. a and ). thus, given the variability observed among natural and experimentally isolated junv strains (as shown in fig. a for the z gene of iv , rumero, xjcl , xj and candid strains), more detailed studies will be necessary in the design of rnai-based therapeutics for successful clinical intervention of human pathogenic arenaviruses. role for a bidentate ribonuclease in the initiation step of rna interference an arenavirus ring (zincbinding) protein binds the oncoprotein promyelocyte leukemia protein (pml) and relocates pml nuclear bodies to the cytoplasm the lymphocytic choriomeningitis virus ring protein z associates with eukaryotic initiation factor e and selectively represses translation in a ring-dependent manner antigenic relationships between attenuated and pathogenic strains of junin virus silencing e a mrna by rna interference inhibits adenovirus replication characterization of the arenavirus ring finger z protein regions 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rna-target recognition and implications for therapeutic approaches an rna-directed nuclease mediates post-transcriptional gene silencing in drosophila cells unlocking the potential of the human genome with rna interference a cellular function for the rna-interference enzyme dicer in the maturation of the let- small temporal rna selective silencing of viral gene expression in hpv-positive human cervical carcinoma cells treated with sirna, a primer of rna interference rna interference: from gene silencing to genespecific therapeutics combination of small interfering rna and lamivudine on inhibition of human b virus replication in hepg . . cells rna interference effectively inhibits mrna accumulation and protein expression of hepatitis c virus core and e genes in human cells transcription and rna replication of tacaribe virus genome and antigenome analogs require n and l proteins: z protein is an inhibitor oh these processes rna interference-based therapeutics: new strategies to fight infectious disease rnai-a powerful tool to unravel hepatitis c virus-host interactions within the infectious life cycle rna interference and antiviral therapy broad-spectrum antiviral activity of small interfering rna targeting the conserved rna termini of lassa virus optimal design and validation of antiviral sirna for targeting hiv- complementary in the supramolecular design of arenavirus and retroviruses revealed by electron cryomicroscopy and image analysis the small ring finger protein z drives arenavirus budding: implications for antiviral strategies myristoylation of the ring finger z protein is essential for arenavirus budding molecular biology of the prototype arenavirus, lymphocytic choriomeningitis virus biochemical and immunological evidence that the kda zinc-binding protein of lymphocytic choriomeningitis virus is a structural component of the virus rna interferencemediated virus clearance from cells both acutely and chronically infected with the prototypic arenavirus lymphocytic choriomeningitis virus junin virus monoclonal antibodies: characterization and cross-reactivity with other arenaviruses lassa virus z protein is a matrix protein sufficient for the release of virus-like particles rnai: double-stranded rna directs the atp-dependent cleavage of mrna at to nucleotide intervals silencing sars-cov spike protein expression in cultured cells by rna interference this work was supported by funding to the group from the consejo nacional de investigaciones científicas y técnicas (conicet), agencia nacional de promoción científica y tecnológica, universidad de buenos aires, and fundación bunge and born, argentina. ccg, las and ebd are members of the research career from con-icet. we are thankful to dr a. sanchez and dr t. ksiazek (cdc, atlanta, ga) for providing junv mabs. key: cord- -ovx fzsg authors: yang, yong-le; liang, qi-zhang; xu, shu-ya; mazing, evgeniia; xu, guo-han; peng, lei; qin, pan; wang, bin; huang, yao-wei title: characterization of a novel bat-hku -like swine enteric alphacoronavirus (seacov) infection in cultured cells and development of a seacov infectious clone date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: ovx fzsg swine enteric alphacoronavirus (seacov), also known as swine acute diarrhea syndrome coronavirus (sads-cov), belongs to the species rhinolophus bat coronavirus hku . herein, we report on the primary characterization of seacov in vitro. four antibodies against the seacov spike, membrane, nucleocapsid and nonstructural protein capable of reacting with viral antigens in seacov-infected vero cells were generated. we established a dna-launched seacov infectious clone based on the cell adapted passage- virus and rescued the recombinant virus with a unique genetic marker in cultured cells. six subgenomic mrnas containing the leader-body junction sites, including a bicistronic mrna encoding the accessory ns a and ns b genes, were experimentally identified in seacov-infected cells. cellular ultrastructural changes induced by seacov infection were visualized by electron microscopy. the availability of the seacov infectious clone and a panel of antibodies against different viral proteins will facilitate further studies on understanding the molecular mechanisms of seacov replication and pathogenesis. swine enteric alphacoronavirus (seacov), also known as swine acute diarrhea syndrome coronavirus (sads-cov), is a novel porcine enteric coronavirus that causes acute vomiting and watery diarrhea in piglets (gong et al., ; pan et al., ; zhou et al., ) . this emerging virus was first isolated from clinically sick animals in commercial swine herds at guangdong province, china during february-may . the mortality rate in less than days old piglets was over %, whereas it dropped to % in piglets older than days . the clinical samples examined by polymerase chain reaction (pcr) or reverse transcription pcr (rt-pcr) during laboratory investigation were negative for the other swine coronaviruses such as porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev), porcine deltacoronavirus (pdcov) and porcine hemagglutinating encephalomyelitis virus (phev), as well as the other known viral pathogens . isolation of the pathogen in african green monkey vero cells resulted in the discovery of seacov , which belongs to the species rhinolophus bat coronavirus hku identified in the same region a decade earlier (lau et al., ) . a retrospective study indicated that the virus had emerged in guangdong since august . the isolated virus was infectious to pigs and cause mild or severe diarrhea symptom when inoculated orally into conventional newborn piglets xu et al., ; zhou et al., ) . nevertheless, as seacov fulfilled the premises of koch's postulates, this was regarded to be the etiologic agent of the epidemic. like other covs, seacov is a single-stranded and positive-sense rna virus in the genus alphacoronavirus (α-covs) of the subfamily coronavirinae of the family coronaviridae. its genome is approximately . kb in size with the gene order of '-orf a/ b (orf ab)-spike (s)-orf -envelope (e)-membrane (m)-nucleocapsid (n)-ns a/ns b- '. seacov shared % nucleotide (nt) sequence identity with the bat cov hku strains and - % nt identity with the hku -derived bat sadsrelated coronavirus (sadsr-cov) strains at the complete genome level zhou et al., ) . interestingly, seacov and other hku -related α-covs possess the unique s genes closely related to the betacoronavirus (β-cov), in a manner similar to those by rodent and asian house shrew α-covs (tsoleridis et al., ; wang et al., wang et al., , b between α-cov and β-cov (lau et al., ; pan et al., ) . the cov genome harbors a few genus-specific accessory genes within the '-part genomic region encoding the four structural proteins (s-e-m-n) . it is found that seacov contains a putative open reading frame (orf), ns a, and a downstream ns b orf (overlapped with ns a) after the n gene at the '-end genome (lau et al., ; pan et al., ) . the ns a is shared by the hku and seacov strains, whereas ns b is only present in the seacov genome . many of cov accessory proteins play some important roles in immune modulation and viral pathogenesis (liu et al., ) . for examples, the severe acute respiratory syndrome coronavirus (sars-cov) orf- a was found to induce necrotic cell death, lysosomal damage and caspase- activation, which largely contribute to the clinical manifestations of sars-cov infection (yue et al., ) . in addition, sars-cov orf and orf b may also be also associated with the virulence. in another newly emerged swine cov, pdcov, its accessory ns protein has been reported to counteract host innate antiviral immune response by inhibiting ifn-β production that interacts with rig-i/mda (fang et al., ) . whether the predicted ns a and ns b of seacov encode functional accessory proteins remain to be confirmed experimentally. discovery of seacov, largely dissimilar to pedv, tgev and pdcov, challenges to the prospects of detection, prevention and control of diarrheal pathogens in swine . it is pivotal to undertake comprehensive investigations on the basic genetics of this emerged enteric cov since very little is known about the molecular virology of seacov. the purpose of this study was to develop seacovspecific antibodies to distinct viral protein as the research tools used to investigate the basic characteristics of seacov infection in vitro. we also aimed to develop a dna-launched reverse genetics system for seacov that will be useful for future studies. . . polyclonal antibodies against four recombinant seacov proteins can react with viral antigens in seacov-infected cells four seacov specific polyclonal antibodies (pabs) against distinct viral protein antigens were generated and validated. two viral genes, seacov n and the nonstructural protein (nsp ) acidic domain (ac) of orf a, were expressed as soluble products in the bacteria; the seacov spike subunit (s ) was expressed in insect cells, secreting into the cultured medium. purified recombinant seacov proteins (n, s and ac) and an antigenic peptide corresponding to the last amino acids (aa) at the carboxyl terminus of the m protein were used to immunize rabbits, respectively, generating four polyclonal sera that were then used to detect viral proteins on seacov-infected vero cells. immunofluorescence assay (ifa) conducted at h post-infection (hpi) using respective pab showed that the four viral antigens (n, m, s or ac) were each expressed in the cytoplasm of the infected cells, with the anti-n and anti-m pabs displaying the higher fluorescence intensity (fig. a) . in contrast, mock-infected controls did not show any positive ifa signals (fig. a) . to determine the intracellular localization and the timing of the viral protein expression with higher magnification, time course analysis of confocal image was performed. vero cells infected with seacov were fixed at , , , and hpi, and labeled with four pab, respectively. perinuclear and cytoplasmic foci were detected by anti-n staining at and hpi, and were distributed throughout the cytoplasm at and hpi, probably reflecting that n protein is associated with sites of viral rna replication in early infection phase and assembled into virions subsequently (fig. b) . anti-ac (nsp ) staining also resulted in detection of perinuclear foci at four time points, indicating localization to the viral replication-transcription complexes (fig. c) , which was similar to the pattern of nsp antibody observed in sars-cov-infected vero cells (prentice et al., ) . confocal microscopy detected discrete cytoplasmic fluorescence signal throughout the cytoplasm with anti-m (fig. d ) and anti-s (fig. e ) as early as hpi. diffuse and more intense fluorescence was observed over time, demonstrating the process of virus assembly by incorporation of m and s proteins into virus particles. the anti-n pab recognized a single band of kda in the lysate of seacov-infected cells but not in control cells at hpi by western blot analysis (fig. f ). the molecular size was consistent with the deduced aa sequence of the n protein but was a little less than the purified products expressed in the bacteria (fig. f) . expression of the m protein with the predicted -kda molecular size was also detected by using anti-m pab in seacov-infected cells (fig. g ). the reactivity of anti-s or anti-ac was less distinct as seen by western blot analysis (data not shown). therefore, all the four seacov pabs can be used for specific detection of seacov infection in the cultured cell by ifa staining, and the anti-n and anti-m pabs can also be used particularly in western blot analysis. the antibodies are available to the research community upon request. genetic manipulation of viral genomes and dissection of the structural and functional relationships of viral genes depend on the development of powerful reverse genetics systems. thus far, the rna polymerases ii-based dna-launched reverse genetics system using a bacterial artificial chromosome (bac) as the backbone vector has been applied to rescue of multiple covs (almazan et al., ) . basically, homogenous rna transcripts are generated from transfected full-length cdna clone in permissive cells to launch virus life cycle. recently, our lab has just developed a novel and efficient method to assemble a fulllength cdna clone of measles virus (~ kb) by using the geneart™ high-order genetic assembly system, without the need for restriction endonucleases, which was used to rescue recombinant measles virus and the derived vaccine candidates . we employed this strategy successfully to assemble the . -kb seacov genomic cdna from the passage- virus ("seacov-p ") by a single step ligation of overlapping fragments into a bac expression vector, resulting in a full-length cdna clone of seacov named psea ( fig. a) . the seacov genomic cdna cassette on psea was engineered with a cytomegalovirus (cmv) promoter and a hepatitis delta virus ribozyme (hdvrz) followed by a bovine growth hormone polyadenylation and termination sequences (bgh) at both termini, respectively. in addition, two silent mutations (a t and g c) in orf were introduced in psea as a genetic marker to distinguish the parental virus seacov-p ( fig. a) . bhk- cells were co-transfected with psea and a helper plasmid expressing the n protein (prk-n) in order to recover the infectious seacov. supernatants from transfected bhk- cells were inoculated onto fresh vero cells at - days post-transfection. seacov-induced cytopathic effects (cpe) were visualized at hpi in inoculated vero cells; viral antigens were detected by ifa using anti-n, anti-m, anti-s or anti-ac to stain cells, confirming the successful recovery of recombinant seacov (rseacov; fig. b ). a region containing the marker from extracellular and intracellular samples of extracted viral rna was amplified and sequenced to determine the retention of the genetic markers in the rescued viruses. the two introduced mutations (tc) were still present in both samples, confirming that the rescued virus originated from the clone psea (fig. c ). there were no other mutations detected in genomic rna of rseacov by genome re-sequencing. we further assessed the morphology of the purified rseacov virions via ultracentrifugation followed by em observation. the virus particles measured - nm in diameter with surface projections (fig. d) , consistent with our previous report of seacov isolation in vero cells . the comparative growth kinetics of rseacov and the parental seacov-p were analyzed by infection of vero cells with the respective virus at the same multiplicity of infection (moi) of . . the infectious virus titers were determined at different time points postinfection ( , , , , , , and hpi) . the result showed that rseacov had the growth kinetics similar to the parental seacov-p ( fig. e ). of note, the maximal rates of seacov-p or rseacov production were from to hpi, suggesting that the exponential release of virus occurred before hpi, which was consistent with detection of n, m, s and ac expression as early as hpi ( fig. b-e) . the single-cycle growth of seacov in vero cells is hence similar to those of mouse hepatitis virus (mhv), sars-cov and pdcov, taking approximately - h (prentice et al., ; qin et al., ) . these data collectively demonstrated that rseacov and its parental virus share the same virological features. to our knowledge, this is the first study describing a seacov/sads-cov infectious clone. previous studies on cov reverse genetics have shown that cov accessory genes such as orf [in tgev , sars-cov (yount et al., ) , pedv (ji et al., ) or human cov nl (donaldson et al., ) ] and the gene [in tgev (ortego et al., ) ] are dispensable for propagation in vitro. the corresponding genes, orf and ns a, are also present in the seacov genome; therefore, we will aim to generate reporter virus expressing luciferase or green fluorescent protein by replacement of orf or ns a with the reporter gene in future studies. coronaviruses can produce multiple sgrnas are produced by discontinuous transcription. each sgrna contains a short ' leader sequence derived from the '-end of the genome and a body sequence from the '-poly (a) stretching to a position in the upstream of each orf encoding a structural or accessory protein (sola et al., ) . the fusion site of the leader and body sequence in each sgrna is termed transcription regulatory sequence (trs). the seacov leader sequence of nt from the '-end to the leader trs was proposed according to the previous report (lau et al., ) ; it was compared with that of another swine α-cov, pedv, indicating an identical leader trs sequence (aactaaa) shared by these two α-covs (huang et al., ) (fig. a) . the existence of all predicted subgenomic mrnas (sgrna; mrna to mrna ) for the expression of s, orf , e, m, n and ns a was investigated further (fig. b) . the leader-body junctions and surrounding regions of all of the putative sgrnas were amplified by rt-pcr. each of the combination of the forward primer (lf) and one of the six reverse primers (s -r, sgorf -r, sge-r, sgm-r, sgn-r and ns a-r) amplified at least one major band of the expected size by agarose gel electrophoresis analysis (fig. c ). the appearance of multiple pcr bands was in line with what was expected, since except for the primers lf and s -r, the other primer combinations could produce larger pcr fragments that seacov-infected or mock-infected vero cells with an anti-n-pab, an anti-m-pab, an anti-s -pab and an anti-ac-pab, respectively (magnification = ×). alexa fluor -conjugated goat anti-rabbit igg (green) was used as the secondary antibody in the ifa. antibody staining merged with nuclear staining using dapi (blue) is also shown. (b-e) time course analysis of n, ac, m or s detection using an olympus confocal microscope. vero cells infected with seacov were fixed at , , , and hpi, and labeled with four pabs, respectively. bar = μm. (f) western blot analysis using cell lysates of seacov-infected or mock-infected vero cells with an anti-n pab. the purified n protein expressed in e.coli was used as the control. (g) western blot analysis using cell lysates of seacov-infected or mock-infected vero cells with an anti-peptide pab specific to m. open arrowheads indicate the detected n or m protein. correspond to the upstream-larger sgrnas. for examples, the primer sgn-r, intended to amplify the leader-body fusion site of mrna , could also amplify those of mrnas to , resulting in detection of five bands (fig. c) . sequencing of individual pcr fragments confirmed that the leader-body junction sequences of sgrnas are identical to the conserved core elements in the intergenic trs (fig. d) . we also noticed that both orfs of ns a and ns b are connected with a body trs in the upstream, implying a bicistronic mrna encoding ns a and ns b (fig. e ). since amplification with the reverse primer ns a-r could not cover the entire ns b, we next determined whether a potential ns b sgrna is present using the leader primer lf and a new reverse primer ns -r corresponding to the '-end of orf b by rt-pcr. a single band of approximately -bp was amplified by optimizing the pcr condition and detected by agarose gel electrophoresis analysis; the other smaller bands were not found (fig. e) . sequence analysis revealed that the trs for this bicistronic sgrna ns was exactly aacuaaa and one nt upstream of the aug start codon of ns a, which is consistent with the prediction (fig. e) . we further expressed and purified the complete ns a or ns b gene in the bacteria. both products were found in the inclusion bodies. however, the resulting anti-ns a or anti-ns b pab did not react with any antigens in seacov-infected cells by ifa and western blot analysis (data not shown) in contrast to the four working seacov pabs. this suggests that ns a and ns b are either, not highly antigenic or the denatured antigens used to generate pabs destroy the native protein structure. development of monoclonal antibodies against ns a and ns b used for experimental validation of the existence of two expression products at the protein level is underway. (e) comparison of growth kinetics between rseacov and the parental seacov-p in vero cells. cells were infected in triplicate with virus at a moi = . . cells were harvested at , , , , , , and hpi, and virus titers (tcid /ml) were determined in triplicate on vero cells. a number of studies on ultrastructural characterization of cov-infected cells in vitro have demonstrated the presence of altered membrane architectures such as the double-membrane vesicles (dmvs), the large virion-containing vacuoles (lvcvs) and the phagosome-like vacuoles during cov replication and morphogenesis (goldsmith et al., ; gosert et al., ; qin et al., ; salanueva et al., ; v'kovski et al., ) . dmvs are membrane structures where viral genomic rna is recognized by the host cell machinery and translated into non-structural proteins (orf ab), assembling into viral replication-transcription complexes (gosert et al., ) , whereas lvcvs are large circular organelles that are thought to originate from golgi compartments expanding to accommodate numerous precursor virions positions of forward (lf) and reverse primers (s -r, sgorf -r, sge-r, sgm-r, sgn-r and ns a-r/ns -r) used for pcr amplification of distinct subgenomic mrnas (sgrnas) are indicated by arrows under the genome. the seven small black boxes at the ' ends of the genomic rna (grna) and sgrnas depict the common leader sequence. genomic and subgenomic rna numbers ( for grna and to for sgrnas) are also indicated. . the other type of membrane structure usually seen is phagosome-like vacuoles or lysosomes containing endoplasmic reticulum (er), small vesicles, damaged mitochondrion and other vesicles. these conserved structures were also observed directly under an electron microscope (em) in seacov-infected vero cells ( fig. a ; hpi) but not in uninfected cells (fig. c) . of note, time course analysis of nsp detection in fig. c likely indicated corresponding locations of the dmvs. since infection of vero cells with either seacov or pedv resulted in indistinguishably cytopathic phenotype, i.e., syncytia formation , the ultrastructural changes in pedv-infected vero cells (at the same moi of . ) were examined under em for comparison of possibly morphological differences. interestingly, pedv appeared to induce a higher number of dmvs and lvcvs in large clusters surrounding the nucleus at hpi and thereafter (fig. b) . a previous study on qualitative and quantitative ultrastructural analysis of membrane rearrangements induced by mhv proposed that cov rna synthesis is dictated by the number of dmvs, whereas an increasing production of viral particles is accommodated by lvcvs from expanding of er-golgi intermediate compartment (ergic)/golgi compartments . it will be interesting to investigate whether synthesis of pedv/seacov rna and assembly of pedv/ seacov virions are correlated with the level of ultrastructural changes in the future. in summary, we generated rabbit antisera against four of the seacov structural and nonstructural proteins and validated their reactivity and use of time course analysis of viral protein expression in seacov-infected vero cells. furthermore, we established a dna-launched reverse genetics system for seacov and rescued the recombinant virus with a unique genetic marker in cultured cells. recombinant seacov had similar growth kinetics to the parental virus. the singlecycle growth of seacov in vero cells was determined to take approximately - h. by rt-pcr analysis, we experimentally identified all proposed seacov sgrnas containing the leader-body junction sites. among six sgrnas, a bicistronic mrna was utilized by the accessory ns a and ns b genes. finally, we characterized the cellular ultrastructural changes induced by seacov infection in vitro. our study develops essential research tools and establishes the basic characteristics of seacov that will facilitate future studies on understanding the molecular mechanisms of seacov replication and pathogenicity. a monkey kidney cell line vero (atcc ccl- ) and a baby hamster kidney fibroblast cell line, bhk- (atcc ccl- ) were grown in dmem supplemented with % fetal bovine serum (fbs) and % antibiotics at °c, respectively. the seacov isolate ch/gd- / at the passage (p ) used in this study was cultured in vero cells. the virus titers were determined by endpoint dilutions as % tissue culture infective dose (tcid ) on vero cells. the control virus pedv (zju/g / strain; genbank accession no. ku ) was also cultured in vero cells as described earlier (ji et al., ; qin et al., ) . vero cells infected by the seacov or pedv (at h postinoculation, hpi) were fixed with . % glutaraldehyde in phosphate buffer ( . m, ph . ) and % oso in phosphate. ultrathin sections were prepared as described previously , stained by uranyl acetate and alkaline lead citrate for - min, and observed using a hitachi model h- tem. polyclonal antibodies (pab) against the spike subunit (anti-s ), membrane (anti-m), nucleocapsid (anti-n) and the nonstructural protein (nsp ) acidic domain (anti-ac) of seacov were produced in rabbits. for generation of anti-m pab, prediction of transmembrane helices of the seacov m protein was first performed using the tmpred software (https://embnet.vital-it.ch/software/tmpred_form.html). the m protein antigenic peptide was predicted as "csdnltendrll-hlv", and synthesized by hua-an biotechnology co., ltd (hangzhou, china). this peptide was purified and used to immunize two new zealand white rabbits and antiserum was harvested at days postimmunization (dpi). anti-s , anti-n and anti-ac pabs of seacov were prepared in-house. briefly, full-length n ( nt, aa,~ kda) or ac ( nt, aa,~ kda) of seacov were expressed with a sixhistidine tag in escherichia coli according to methods described previously (huang et al., ) , whereas seacov-s ( nt, aa, kda) with a six-histidine tag was expressed by baculovirus system in sf insect cells as described previously (wang et al., a) . the purified proteins were used to immunize rabbits, and antisera were harvested at dpi, respectively. total rna from seacov-infected vero cell was extracted using trizol reagent (invitrogen) and then reverse-transcribed with a superscript ii reverse transcriptase (invitrogen) using oligo-dt (promega) as the reverse primer according to the manufacturer's instructions. the forward primer lf ( '-atagagtccttatcttttt- ') and six gene specific reverse primers, s -r ( '-caatggcatttctgtg tacctctc- '), sgorf -r ( '-agtaatctgcttacaacagc- '), sge-r ( '-agacattaattatggggcat- '), sgm-r ( '-gttcgcgttctgcga taaag- '), sgn-r ( '-atctgcgtgaggaccagtac- '), ns a-r ( '-aatctgcaaaatctgccaac- '), were designed for amplification of all seacov subgenomic mrnas (fig. a ) from the obtained cdna with a taq dna polymerase (transgen, beijing, china) in a total volume of μl by pcr. the pcr condition was set at cycles of °c for s, °c for s, °c for min with an initial denaturing of the template dna at °c for min and a final extension at °c for min. the resulting pcr fragments were analyzed on a % agarose gel (fig. b) and then subcloned into a peasy-t vector (transgen, beijing, china) followed by sanger sequencing. for amplification of the subgenomic mrna containing the entire ns a/ns b, the reverse primer ns -r ( '-ttacgtgcttaccattgtgt- ') was used, and the pcr extension time was shortened to s. analysis of dna sequences was performed using the lasergene package (dnastar inc., madison, wi). the expression vector, designated as psb μ, used to construct a fulllength seacov cdna clone, was based on a bac backbone vector psmart-bac-bamhi (copyright v . bac cloning kits, lucigen). this psmart-bac vector was modified to insert a yeast replication origin ( μ) from the plasmid pyes (invitrogen), a cytomegalovirus (cmv) promoter from the plasmid pcdna (invitrogen), a hepatitis delta virus ribozyme (hdvrz) sequence from a prrsv (porcine reproductive and respiratory syndrome virus) infectious clone ptri- rz-pgxg (huang et al., ) , and a bovine growth hormone (bgh) polyadenylation and terminator from the plasmid pcdna (invitrogen) by several rounds of amplification and "in-fusion" pcr according to our previous publication . the primer sequences and approaches used in the pcr assays are available upon request. the full-length consensus sequence of seacov-p ( , nt) was determined as described previously . briefly, a total of overlapping fragments covering the entire genome was amplified by rt-pcr using the q high-fidelity ×master mix (new england biolabs, usa). pcr products were purified and cloned into a peasy-blunt vector (transgen, beijing, china) . for each amplicon, five individual clones were sequenced to validate the consensus sequence. to create a -nt genetic marker on the orf gene of the infectious clone, two point mutations, a to t, and g to c at nucleotide positions - , corresponding to the seacov-p genome, were generated on the fragment s- by fusion pcr (fig. a) . subsequently, all fragments identical to the consensus sequence together with the mutated s- fragment were re-amplified from each clone with primers listed in table . it was then assembled into the expression vector (psb μ) between the cmv promoter and the hdvrz+bgh element, using the geneart™ high-order genetic assembly system according to the manufacturer's manual, to create a dna-launched seacov fulllength cdna clone, psea (fig. ) . the plasmid psea is available to the research community upon request. the sequence encoding the fulllength seacov nucleocapsid gene was amplified and inserted into a prk eukaryotic expression vector containing a flag-tag at its c terminus to construct prk-n-flag as a helper plasmid for rescuing the infectious clone. the plasmid psea was purified from the e. coli dh b strain using qiaprep miniprep kit (qiagen) and quantified by a nanodrop spectrophotometry. bhk- cells were seeded at × per well of a sixwell plate and grown until - % confluence before transfection. one microgram each of psea and prk-n-flag were co-transfected into the cells using lipofectamine (invitrogen) according to the manufacturer's protocol. transfected cells were cultured for - days. the supernatant was collected and passaged onto fresh vero cells on -well plates and cultured for days before the detection of viral protein expression by ifa. the recombinant seacov rescued from the psea infectious clone was named rseacov. the rseacov titers were determined by endpoint dilutions as tcid . viral particles in the supernatants from rseacov-infected cell cultures were negatively stained and examined under tem. a . -kb dna fragment harboring the introduced mutations in the orf gene was amplified by rt-pcr using primers tf ( '-tactggatgttgtggcatgt- ') and tr ( '-ttccacttaaaatcgtcaga- '). the amplicons were sequenced to affirm that rseacov contained the desired mutations. seacov-infected or rseacov-infected cells were washed twice with pbs, fixed with % paraformaldehyde in pbs for min and then permeabilized with . % triton x- for min. anti-n, anti-m, anti-s or anti-ac pab, each at a : dilution in pbs, was added over the cells and incubated for h at °c. cells were then washed thrice with pbs and alexa fluor -labeled goat anti-rabbit igg (thermo fisher scientific) at a : dilution was then added. after min of incubation at °c, the cells were again washed thrice with pbs followed by ', -diamidino- -phenylindole (dapi) staining, and were visualized under a fluorescence microscope (dmi b, leica, germany). for time course analysis of detection of n, m, s or ac, fluorescent images were obtained with a confocal laser scanning microscope (fluoviewver fv -ix ; olympus, japan). for western blot analysis, seacov-infected cells were lysed in lysis buffer ( mm tris-hcl, mm nacl, mm naf, mm na vo , mm β-glycerophosphate, % np , and protease cocktail [biotool, houston, tx]). samples were resolved on sds-page and transferred onto polyvinylidene difluoride (pvdf) membrane that was subsequently blocked with tris-buffered saline (tbs) containing % bovine serum albumin (bsa) overnight at °c. proteins were detected using the anti-n pab or anti-m pab at : dilution followed by incubation with horseradish peroxidase (hrp)-conjugated anti-rabbit igg ( : dilution; thermo fisher scientific). the consensus sequence of seacov-p used for construction of the infectious clone has been deposited in genbank under accession no. mk . natural science foundation of china ( ), and the fundamental research funds for the central universities of china ( fza ). we thank the staff in the shared experimental platform for core instruments, college of animal science, zhejiang university for assistance with analysis of confocal microscopy. coronavirus reverse genetic systems: infectious clones and replicons systematic assembly of a full-length infectious clone of human coronavirus nl porcine deltacoronavirus accessory protein ns antagonizes interferon beta production by interfering with the binding of rig-i/mda to 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causing watery diarrhoea and high mortality in newborn piglets severe acute respiratory syndrome coronavirus groupspecific open reading frames encode nonessential functions for replication in cell cultures and mice sars-coronavirus open reading frame- a drives multimodal necrotic cell death retrospective detection and phylogenetic analysis of swine acute diarrhoea syndrome coronavirus in pigs in southern china this work was supported by the national key research and development program of china ( yfd ), the national key: cord- -njb x authors: khan, mohsin; santhosh, s.r.; tiwari, mugdha; lakshmana rao, p.v.; parida, manmohan title: assessment of in vitro prophylactic and therapeutic efficacy of chloroquine against chikungunya virus in vero cells date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: njb x the resurgence of chikungunya virus (chikv) in the form of unprecedented and explosive epidemics in india and the indian ocean islands after a gap of years is a major public health concern. currently, there is no specific therapy available to treat chikv infection. in the present study, the in vitro prophylactic and therapeutic effects of chloroquine on chikv replication in vero cells were investigated. inhibitory effects were observed when chloroquine was administered pre‐infection, post‐infection, and concurrent with infection, suggesting that chloroquine has prophylactic and therapeutic potential. the inhibitory effects were confirmed by performing a plaque reduction neutralization test (prnt), real‐time reverse transcriptase (rt)‐pcr analysis of viral rna levels, and cell viability assays. chloroquine diminished chikv infection in a dose‐dependent manner, with an effective concentration range of – µm. concurrent addition of drug with virus, or treatment of cells prior to infection drastically reduced virus infectivity and viral genome copy number by ≥ . %. the maximum inhibitory effect of chloroquine was observed within – hr post‐infection (hpi), and treatment was ineffective once the virus successfully passed through the early stages of infection. the mechanism of inhibition of virus activity by chloroquine involved impaired endosomal‐mediated virus entry during early stages of virus replication, most likely through the prevention of endocytosis and/or endosomal acidification, based on a comparative evaluation using ammonium chloride, a known lysosomotropic agent. j. med. virol. : – , . © wiley‐liss, inc. the re-emergence of chikungunya virus (chikv) in many parts of the world, with associated severe clinical features, is a significant public health concern. since , chikv infection has assumed epidemic proportions in asia and sub-saharan africa. several outbreaks of chikv fever occurred in , and virus was disseminated among the populations of several islands in the indian ocean (the comoros, mauritius, seychelles, madagascar, la reunion) prior to outbreaks in india, where an estimated . million cases have been reported [charell et al., ; mavalankar et al., ; pialoux et al., ] . recent cases of chikv infection in europe and italy have occurred as a result of travel to and from infected areas [rezza et al., ] . chikv is an arthropod-borne virus of the alphavirus genus of the togaviridae family. it is transmitted primarily to humans by aedes aegypti and aedes albopictus mosquitoes. like other alphaviruses, the genome of chikv consists of a linear, positive-stranded rna molecule of $ . kb [jupp and mcintosh, ]. chikv causes an acute illness characterized by fever, headache, skin rash, vomiting, myalgia, and polyarthralgia [jupp and mcintosh, ]. there is no effective treatment or licensed vaccine available for the clinical management of chiky infection. in the absence of an effective vaccine and mosquito control measures, it is necessary to seek effective anti-viral drugs for immediate relief for affected patients and to reduce viremia. the therapeutic application of small interfering rna (si-rna) for the inhibition of chikv replication has achieved limited success . chloroquine is an effective anti-malarial drug in areas where resistance has not been established. increasingly, chloroquine is being applied to the clinical management of viral diseases [savarino et al., [savarino et al., , . chloroquine as an effective anti-viral therapeutic for the clinical management of viral diseases was first established in the s for hiv- infection [savarino, ] . anti-viral effects of chloroquine against sars-cov, hiv type and hepatitis b virus have also been reported [kouroumalis and koskinas, ; tsai et al., ; vincent et al., ] , and the use of chloroquine as a therapeutic for hiv- infection is currently being evaluated in clinical trials [savarino et al., ] . in light of its availability and cost, and the fact that it is well tolerated, chloroquine offers promise as an anti-viral and immunomodulatory agent for the treatment of emerging viral diseases [keyaerts et al., ] . increased virulence of chikv as a result of evolutionary adaptation during chikungunya outbreaks has been reported [schuffenecker et al., ; santhosh et al., ] . thus, it has become increasingly important to develop effective therapeutic approaches for the treatment of chikv infection. the goal of the current study was to evaluate the doseand time-dependent effects of chloroquine on chikv replication, and to elucidate the mechanism of viral inhibition in vero cells. vero cells were obtained from the national centre for cell sciences (nccs), pune, india, and maintained in eagles minimal essential medium (emem) supplemented with . g sodium bicarbonate/l, % heatinactivated fetal bovine serum (fbs), mm l-glutamine, and u of gentamycin. the chivk isolate drde- , which belongs to the ecs african genotype, was used in the present study . increasing concentrations of chloroquine ( , , and mm) were added to cultured vero cells after determining the maximum non-toxic dose. the cells were treated with chloroquine as follows: for the pre-treatment group, the drug was added to the cells hr prior to infection; for the concurrent treatment group, the drug and chikv were administered at the same time; for the post-treatment group, the drug was added at different points from - hr following infection of cells with chikv. in the pre-treatment mode, chloroquine was removed by washing the cells before infection. in the concurrent and post-treatment modes, the drug was maintained in culture until the supernatants were harvested. cells were seeded in a cm cell culture flask at a density of  cells/ml (  cells per flask) and then incubated for hr. cells were infected with chikv at a multiplicity of infection (m.o.i.) of . . drug was administered to the three different treatment groups ( hr pre-treatment, concurrent treatment, and post-treatment hpi). infection was allowed to proceed for hr, at which time cells were scraped and virus was released into the supernatant by freeze thawing the cells three times. cell pellets were removed by centrifugation at , g for min. virus yield was determined by the plaque assay. cells were seeded on -well culture plates (greiner bio-one, solingen, germany) at a density of  cells/ well and allowed to grow to % confluency. the medium was discarded and the cell monolayer was infected with chikv ( pfu/well). seeded cells were treated with drug hr before infection, concurrent with infection, or hpi. after a period of hr to allow for virus adsorption, cells were overlaid with an overlay medium containing . % methylcellulose, % fcs, and the appropriate concentration of drug. after hr, the overlay medium was removed and the infected cell monolayer was fixed in % pbs-formaldehyde. virus plaques that formed on vero cells were visualized by staining with % crystal violet. percent inhibition was determined relative to untreated control cells. anti-viral activity was assessed by performing cell viability assays on cells that had been infected with chikv in the presence of various concentrations of ammonium chloride, ribavirin, or chloroquine. the number of viable cells was quantified hpi by neutral red dye uptake assay [finter, ] . a selectivity index for each test compound for the pre-treatment, concurrent, and post-treatment ( hpi) groups was determined as the ratio of the concentration of test compound required to reduce cell viability by % (cc ) to the concentration required to inhibit virus infectivity by % as compared to control cells (ic ). vero cell monolayers cultured in cm flasks were infected with chikv (m.o.i. of . ) to % confluence. increasing concentrations of drug ( , , and mm) were added to all treatment groups (pre-treatment, concurrent, and post-treatment hpi). infection was allowed to proceed for hr, at which time ml of culture supernatant was drawn from each treatment group in triplicate and then pooled. genomic viral rna was extracted from ml of pooled supernatant using a qiaamp viral rnamini kit (qiagen, hilden, germany), according to the manufacturer's protocol. the total copy number of chikv genomes was analyzed by sybr green i-based one-step real-time quantitative rt-pcr, as previously described . a region of the envelope e gene was amplified using the following specific primers: -acgcaattgagcgaag-cac- (forward), -ctgaagacattggccccac- (reverse). real-time rt-pcr was performed using the mx p quantitative pcr system (stratagene, la jolla, ca). test samples were analyzed following optimization with rna standards using the brilliant sybr green single-step qrt-pcr master mix (stratagene). after amplification, a melting curve analysis was performed to verify the authenticity of the amplified product according to its specific melting temperature (t m ) using the melting curve analysis software of the mx system. analysis of relative cycle threshold (c t ) values was performed and the overall reduction in genome copy number was calculated by plotting c t versus genome copy number. subconfluent monolayers of vero cells in -well plates were infected with chikv in duplicate, and then treated with chloroquine at a concentration of mm for increasing periods of time post-infection ( to hpi). supernatants were collected at each time point and viral load was determined by plaque titration to assess chikv growth kinetics. the effect of chloroquine on virus internalization was assessed by the immunofluorescence test (ift). cultured vero cells were infected with chikv in the presence or absence of drug and infection was allowed to proceed for hr. cells were washed five times with pbs, and then fixed using chilled methanol. cells were permeabilized using . % triton-x for the detection of intracellular virus. fixed cells were incubated with rabbit anti-chikv hyperimmune serum ( : , dilution) followed by fitc-conjugated anti-rabbit igg (sigma, st. louis, mo) ( : ). cells were washed and then observed using a carl-zeiss aximot (thuringia, germany) microscope, which was equipped for incident illumination with a narrow band filter combination specific for fitc. the mechanism of inhibition of chikv activity by chloroquine was assessed by comparing the effects of chloroquine to those of a known lysomotropic agent (ammonium chloride) that interferes with early stages of infection, and a standard anti-viral compound (ribavirin) that inhibits virus replication during late stages of infection. confluent monolayers of vero cells were infected at an m.o.i of . with chikv, and then treated with appropriate concentrations of ammonium chloride, ribavirin and chloroquine hr before and hr after chikv infection. in the case of pre-treatment, compounds were removed by washing before infection. cell viability was measured hpi by the neutral red dye uptake assay, as described above. prior to screening, we determined the maximum nontoxic dose of chloroquine for vero cells. a concentration of mm chloroquine was non-toxic to vero cells. the growth kinetics of chikv in vero cells at different multiplicities of infection was also determined to establish an appropriate time line for harvesting and subsequent analysis of viral activity. the optimum virus yield following infection with a titer of  pfu/ml was obtained hpi. to determine the anti-chikv activity of chloroquine, we analyzed virus yield in vero cells treated with drug as compared to untreated infected control cells. there was a substantial decrease in viral titer when cells were pre-treated with several different concentrations of chloroquine. concurrent treatment and post-treatment ( hpi) with chloroquine also inhibited chikv infection at higher concentrations. viral titer was reduced nearly % by mm chloroquine, as indicated by the - log decrease in virus yield in all treatment groups. these results provided substantial evidence of the anti-chikv activity of chloroquine (fig. a) . anti-chikv activity was also evaluated by plaque reduction assay. in the presence of mm chloroquine, plaque formation was inhibited %, %, and % in the pre-treatment, concurrent, and post-treatment ( hpi) groups, respectively (fig. b) . we next evaluated the cell viability of infected vero cells in the presence of different concentrations of chloroquine by neutral red dye uptake assay. based on the optical density at nm (od ) of treated and untreated cells, ic , ic , and a selectivity index were calculated (table i) . pre-treatment with chloroquine was the most effective anti-chikv strategy, as indicated by a nearly . -fold higher selectivity index for the pre-treatment group as compared to the post-treatment group. we analyzed viral genome copy number following infection with chikv using real-time rt-pcr. viral rna was isolated from the culture supernatants of chloroquine-treated and -untreated cells, and then amplified using e gene-specific primers, as described in materials and methods section. the inhibition of chikv activity by chloroquine was evaluated by comparing c t values obtained for each experimental condition, and the specificity of the amplified product was analyzed by t m curve analysis. as depicted in figure a -d, the amplification curves revealed higher c t values for the pre-treatment, concurrent, and posttreatment groups at all concentrations of chloroquine as compared to infected cells. these results indicated that chloroquine treatment reduces viral rna load, thereby inhibiting chikv replication. the c t values for all treatment groups and concentrations of chloroquine are shown in table ii . in addition to relative c t values, we also determined the absolute values for genome copy number using a standard curve, and observed an overall - log reduction in viral load in a dose-dependent manner (fig. e) . to determine whether chloroquine inhibited chikv internalization, we analyzed the location of intracellular viral antigens by ift. infected vero cells that were treated with chloroquine exhibited lower levels of fluorescence intensity as compared to infected cells and this decrease in fluorescence intensity was dose dependent (fig. ) . as compared to infected cells, chloroquine pre-treated infected cells exhibited lower fluorescence intensity, and in the presence of mm chloroquine, fluorescent cells were undetectable, which indicated a near complete inhibition of virus internalization. we carried out a time course analysis to determine the kinetics of viral inhibition by chloroquine, and found that the anti-viral effects of chloroquine decreased significantly when the drug was added later than hpi (fig. ) . the addition of chloroquine during the early stages of viral infection ( - hpi) significantly affected viral yield, but at later stages, the drug was ineffective, suggesting that the mechanism of inhibition of chikv by chloroquine involves the early stages of virus replication. to begin to investigate the putative mechanism of action of chloroquine, we compared the effects of chloroquine to those of the anti-viral compounds ribavirin and ammonium chloride. ammonium chloride was effective against chikv only when it was added prior to infection, and did not protect cells when added hpi, based on cell viability (fig. a) . in contrast, ribavirin was effective against chikv infection only when it was added at the time of infection or after infection, but did not protect cells when it was added prior to infection and then removed by washing (fig. b) . thus, the pattern of protection by chloroquine was similar to that of ammonium chloride, in that pretreatment of cells inhibited virus replication, but there was no inhibitory effect after hpi. (fig. c ). currently, there is no specific anti-viral treatment for chikv infection. we demonstrated that chloroquine is an effective anti-viral agent against chikv infection in vero cells in culture, thus, demonstrating the in vitro prophylactic and therapeutic potential of chloroquine in inhibiting chivk infection. chloroquine treatment significantly reduced virus yield, and reduced plaque forming ability by more than % (based on the plaque forming activity of pfu of virus) (fig. b) . there was also a significant reduction in viral rna copy number, based on real-time rt-pcr analysis (fig. ) , providing strong evidence of the therapeutic potential of chloroquine in inhibiting chikv replication. in cell viability assays, chloroquine treatment provided near complete protection of vero cells against chikv infection, which provided further evidence of the anti-viral potential of this drug. previously, chloroquine was suggested as an effective agent against viral infection [savarino et al., ] . the data obtained from the current study indicate j. med. virol. doi . /jmv real-time rt-pcr analysis of chivk genome copy number. amplification plots (fluorescence vs. cycle) depicting the relative abundance of chikv rna in the supernatants of infected cells treated with , , and mm chloroquine. the specificity of the amplified products was analyzed by t m curve analysis (a). amplification plots for cells treated with chloroquine hr before (b), concurrently (c), and hpi (d). for all treatment groups, the fold-reduction in genome copy number was calculated and plotted against chloroquine concentration (e). [color figure can be viewed in the online issue, which is available at www.interscience. wiley.com.] that chloroquine is effective against the novel ecsa genotype of chivk that has caused several recent explosive and unprecedented epidemics. chloroquine is a weak base that targets acid vesicles, leading to the dysfunction of several proteins. chloroquine has been shown to inhibit protease activity and affect dna synthesis [cassell et al., ] . however, our results suggest that the anti-viral activity of chloroquine is not associated with these previously reported activities, since chikv infection was unaffected when the drug was added during late stages of viral infection. thus, in the case of pre-treatment, the presence of chloroquine might not be essential for viral inhibition, whereas chloroquine is necessary at least up to hpi to significantly inhibit virus yield. the addition of chloroquine at hpi had no effect on viral replication. our results suggest that chloroquine is effective at early stages of viral infection, and that the effects are doseand time-dependent. the mechanism of action of chloroquine appears to depend on the mode of treatment. in pre-treatment mode, cells were rendered refractory to chikv infec- [vincent et al., ] . a similar mechanism may be responsible for the inhibition of chikv infection by chloroquine. in the case of alphaviruses like sindbis virus (sinv) and semilink forest virus (sfv), conformational changes in the viral envelope glycoprotein and subsequent viral fusion are mediated by clathrinmediated endocytosis by the target cell and the low ph of the endosomal compartment [detulleo and kirchhausen, ]. it has been reported that a low endosomal ph is also required for chikv entry into cells [sourisseau et al., ] . in the case of concurrent treatment and post-treatment ( hpi), rapid elevation of endosomal ph and abrogation of virus-endosome fusion might be the primary mechanism by which virus infectivity is inhibited by chloroquine. the kinetics of inhibition based on a time course analysis clearly imply that the anti-viral effects of chloroquine decline substantially when the drug is added later than hpi (fig. ) . in the post-treatment group, the addition of chloroquine at an early stage ( - hpi) of infection had a marked effect on virus yield, whereas late stage addition ( - hpi) was ineffective. the ic of chloroquine for inhibiting chikv in vitro is similar to the plasma concentration of chloroquine reached during the treatment of acute malaria [charmot and coulaud, ] . thus, chloroquine might inhibit chikv infection and its subsequent dissemination. the effect of chloroquine on the internalization of chikv was investigated by immunofluorescence analysis of intracellular viral antigen. infected vero cells treated with chloroquine exhibited markedly lower levels of fluorescence intensity as compared to infected cells, and this effect was dose dependent with complete inhibition at higher concentrations of chloroquine (fig. ) . the results of ift also supported the finding that pre-treatment of cells with or mm chloroquine was more effective than concurrent treatment and posttreatment ( hpi), which were effective to a lesser extent at higher concentrations of chloroquine. these results suggest that chloroquine treatment prevents or delays virus internalization. in order to gain an understanding of the mechanism of action of chloroquine, we compared the effects of chloroquine to those of the well-known anti-viral compounds ribavirin and ammonium chloride. ribavirin is an anti-viral compound that inhibits a number of viruses, including chikv, and acts at late stages of viral infection [gilbert and knight, ] . ammonium chloride is a lysomotropic agent that blocks early stages of infection, for example, endosome-mediated virus entry, and has no effect during later stages of infection [cassell et al., ] . ammonium chloride was effective against chikv when cells were pre-treated ( hr before), and retained its anti-viral activity even when it was removed prior to infection. however, administration of ammonium chloride hpi did not protect cells from chikv infection (fig. a) . in contrast, ribavirin was effective against chikv infection only when administered after infection. no inhibitory effect was observed when cells were pre-treated with ribavirin followed by removal of the drug before infection (fig. b) . thus, chloroquine (fig. c) and ammonium chloride exhibited similar patterns of inhibition of chikv propagation, suggesting that chloroquine might also target the early stages of chikv infection. in summary, the results of the current study suggest that chloroquine inhibits chikv infection in vero cells though a mechanism that involves the early stages of infection. the fact that chloroquine exerts its anti-viral effects in all the three modes of treatment (pre-treatment, concurrent, and post-treatment) suggests that it has prophylactic and therapeutic potential. chloroquine blocks the production of proinflammatory cytokines and the proliferation of monocytes, macrophages, and lymphocytes. thus, it represents a potential drug for the treatment of some of the symptoms of chikungunya disease. since immunopathological factors might play an important role in chikv infection, it would be relevant to explore the effects of chloroquine on the inflammatory response to chikv infection. effects of lysosomotropic weak bases on infection of bhk- cells by sindbis virus chikungunya outbreaksthe globalization of vector borne diseases treatment of plasmodium falciparum malaria in africa (except cerebral malaria) east central south african genotype as the causative agent in reemergence of chikungunya outbreak in india rna interference mediated inhibition of chikungunya virus replication in mammalian cells the clathrin endocytic pathway in viral infection dye uptake methods for assessing viral cytopathogenicity and their application to interferon assays biochemistry and clinical application of ribavirin chikungunya virus disease in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine treatment of chronic active hepatitis b (cah b) with chloroquine: a preliminary report chikungunya epidemic in india: a major public-health disaster chikungunya, an epidemic arbovirosis infection with chikungunya virus in italy: an outbreak in a temperate region development and evaluation of sybr green i based one step real time rt-pcr assay for detection and quantification of chikungunya virus comparative full genome analysis revealed a v shift in indian chikungunya virus isolates expanding the frontiers of existing antiviral drugs: possible effects of hiv- protease inhibitors against sars and avian influenza effects of chloroquine on viral infections: an old drug against today's diseases? new insights into the antiviral effects of chloroquine genome microevolution of chikungunya viruses causing the indian ocean outbreak characterization of reemerging chikungunya virus inhibition of human immunodeficiency virus infectivity by chloroquine chloroquine is a potent inhibitor of sars coronavirus infection and spread the authors are thankful to dr. r. vijayaraghavan, director, defence research and development establishment, ministry of defence, government of india, for his support, constant inspiration and for providing the necessary facilities for this study. key: cord- - xjdryoq authors: scholte, florine e. m.; tas, ali; martina, byron e. e.; cordioli, paolo; narayanan, krishna; makino, shinji; snijder, eric j.; van hemert, martijn j. title: characterization of synthetic chikungunya viruses based on the consensus sequence of recent e - v isolates date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: xjdryoq chikungunya virus (chikv) is a mosquito-borne alphavirus that re-emerged in and has caused massive outbreaks in recent years. the lack of a licensed vaccine or treatment options emphasize the need to obtain more insight into the viral life cycle and chikv-host interactions. infectious cdna clones are important tools for such studies, and for mechanism of action studies on antiviral compounds. existing chikv cdna clones are based on a single genome from an individual clinical isolate, which is expected to have evolved specific characteristics in response to the host environment, and possibly also during subsequent cell culture passaging. to obtain a virus expected to have the general characteristics of the recent e - v chikv isolates, we have constructed a new chikv full-length cdna clone, chikv ls , based on the consensus sequence of their aligned genomes. here we report the characterization of this synthetic virus and a green fluorescent protein-expressing variant (chikv ls -gfp). their characteristics were compared to those of natural strain ita -ra , which was isolated during the outbreak in italy. in cell culture the synthetic viruses displayed phenotypes comparable to the natural isolate, and in a mouse model they caused lethal infections that were indistinguishable from infections with a natural strain. compared to ita -ra and clinical isolate nl / , the synthetic viruses displayed similar sensitivities to several antiviral compounds. -deaza-adenosine was identified as a new inhibitor of chikv replication. cyclosporin a had no effect on chikv replication, suggesting that cyclophilins -opposite to what was found for other +rna viruses- do not play an essential role in chikv replication. the characterization of the consensus sequence-based synthetic viruses and their comparison to natural isolates demonstrated that chikv ls and ls -gfp are suitable and representative tools to study chikv-host interactions, screen for antiviral compounds and unravel their mode of action. chikungunya virus (chikv) re-emerged in and has caused unprecedented outbreaks in asia and africa since . the estimated number of cases exceeds million and over a thousand infected travelers have returned to europe and the usa since [ , ] . chikv generally causes a fever that resolves within several days, a maculopapular rash, and a characteristic arthralgia that can be extremely painful and may persist for months. during the recent outbreaks also more severe clinical manifestations have been reported occasionally, such as neurological complications and even deaths, usually in the elderly, patients with underlying conditions, and newborns [ , ] . a licensed vaccine or specific antiviral therapy are currently not available. chikv is an alphavirus with an . kb positive-stranded rna genome that contains two open reading frames (orfs). the orf encodes the nonstructural polyproteins p and p . the latter results from translational read-through of an opal termination codon that is present at the end of the nonstructural protein (nsp) coding sequence of most chikv isolates. assuming that chikv follows the typical alphavirus life cycle, proteolytic processing of the nonstructural polyproteins by the protease domain in nsp will ultimately lead to the release of nsp , nsp , nsp , and nsp . these nsps and their precursors possess a variety of functions and the enzymatic activities, including protease, helicase, methyltransferase, and rna-dependent rna polymerase (rdrp) activity that drive chikv replication [ ] . in addition to replication of its genomic rna, chikv also transcribes a subgenomic (sg) rna encoding a precursor polyprotein that is processed by viral and cellular proteases into the structural proteins c, e , e , k and e . chikv nsps willpresumably together with host factors -assemble into replication and transcription complexes (rtcs) that associate with membrane structures derived from the plasma membrane and/or endosomes, as observed for other alphaviruses [ ] [ ] [ ] . the chikv strains that emerged during the - outbreaks had acquired a mutation (a v) in the e envelope glycoprotein, which facilitated transmission of the virus via a new vector, the asian tiger mosquito aedes albopictus, and consequently dramatically increased the epidemic potential of chikv [ , ] . later studies suggested that recent indian and indian ocean epidemics have emerged separately as the result of at least three independent events, and that convergent evolution of east-central-south african lineage strains in different geographical regions ultimately led to the emergence of strains with the a v substitution in e [ ] [ ] [ ] [ ] . more recently, other amino acid positions and epistatic interactions were also shown to play an important role in the emergence of these new chikv variants, which are now even replacing endemic strains that have been circulating in asia for decades [ ] . aedes albopictus also thrives in more temperate climates and its geographical distribution has rapidly expanded. over the past decades, parts of southern europe and large areas of the usa have been invaded by this mosquito, providing imported cases of chikv with a competent mosquito vector, thus paving the road for outbreaks in nonendemic-areas such as the usa and europe. indeed, autochthonous infections have been reported from italy in and france in [ , ] . the recent and ongoing chikv outbreaks are characterized by their rapid geographical spread, high numbers of infected people and high morbidity, emphasizing the need to gain more insight into the replicative cycle of this important human pathogen. infectious cdna clones of viruses have become invaluable tools that allow reverse genetics studies to elucidate the contribution of specific amino acids or rna structures to viraemia, virulence, antigenicity, replication kinetics, interactions with host factors, adaptation to new vectors, and many other aspects of the viral life cycle. the use of cdna clones is also instrumental in mechanism of action studies to pinpoint the viral target of antiviral compounds by selecting for and genotyping compound-resistant viruses, followed by reverse engineering of the identified mutations to assess their individual phenotypic contribution to resistance. finally, the generation of cdna clones of reporter viruses, like those expressing green fluorescent protein (gfp), greatly facilitates high throughput screening, e.g. for antiviral compounds or host factors that affect replication. several chikv cdna clones have been constructed in the past, which -except for the west african lineage strain strain that was isolated from a mosquito -were all based on clinical isolates from infected humans [ ] [ ] [ ] [ ] [ ] [ ] [ ] . each natural isolate is expected to have evolved its own specific characteristics in terms of sequence, virulence and virus-host interactions as a result of specific selective pressures within the infected host (tissue) and possibly also during subsequent passaging in cell culture. intrahost evolution and quasispecies diversity is expected to be substantial, especially compared to the relatively low level of interhost variation when the consensus sequences of chikv genomes isolated from different hosts are aligned. the low level of interhost variation is a typical trait of arboviruses, due to evolutionary constraints imposed by the alternating replication in vertebrate and arthropod hosts. a recent study on the distantly related ross river virus indeed reported a high level of intrahost diversity [ ] . the existing chikv molecular clones can be considered to represent a single individual genome (or fragments of several individual genomes) out of the whole spectrum of viruses present in the chikv quasispecies population that has been shaped by intrahost evolution and probably a complex set of environmental factors. in contrast, most deposited chikv genome sequences represent the consensus (or master sequence) of a viral quasispecies population. to obtain a virus that -in terms of virulence, sensitivity to antiviral compounds, and chikv-host interactions -is expected to have the general characteristics of the e - v chikv strains that were circulating during the - outbreaks, we have constructed a completely synthetic chikv cdna clone based on the consensus sequence of the aligned genomes of these recent isolates. this new infectious clone, chikv ls (leiden synthetic ), and a variant that expresses the egfp reporter gene under control of a duplicated subgenomic promoter (chikv ls -gfp), were created by custom dna synthesis. the properties and replicative cycle of the new synthetic viruses were characterized in detail, and comparison with a field isolate (ita -ra ) from the chikv outbreak in italy demonstrated that they have similar characteristics. the sensitivity of ls to a number of antiviral compounds was compared to those of ita -ra and clinical isolate nl / . all compounds tested had a similar antiviral activity against ls and the natural isolates. these experiments also identified -deaza-adenosine as a novel inhibitor of chikv replication. this study describes a detailed characterization of the chikv replication cycle at the molecular level and demonstrates that a new synthetic infectious clonederived virus is a useful and representative tool to gain more insight into the replicative cycle of chikv, its interactions with the host, and the mode of action of antiviral compounds, which should aid in the development of antiviral strategies against this important human pathogen. vero e , ae. albopictus c / [ ] and /ace cells [ ] were maintained in dulbecco's modified eagle's medium (dmem; lonza), supplemented with % fetal calf serum (fcs; paa), mm l-glutamine, iu/ml of penicillin and mg/ml of streptomycin. /ace cells were grown in the presence of mg/ml blasticidin (paa) and c / medium was supplemented with non-essential amino acids (lonza). bhk- cells were cultured in glasgow's modified eagles medium (gibco) supplemented with . % fcs, mm hepes ph . , % tryptose phosphate broth (gibco), and antibiotics. the mammalian cell lines were grown at uc and c / cells at uc in % co . chikv strain ita -ra (genbank accession number eu ) was isolated from ae. albopictus during the outbreak in ravenna, italy, and was passaged twice on bhk- cells. chikv nl / (genbank kc ) was isolated at the erasmus medical center in rotterdam from the serum of an infected traveler that returned from indonesia and was passaged twice on vero cells. working stocks of chikv were routinely produced in vero e cells at uc, typically yielding titers of , pfu/ml. infections were performed in eagle's minimal essential medium (emem; lonza) with mm hepes (lonza) supplemented with % fcs, l-glutamine, and antibiotics. after h, the inoculum was replaced with fresh culture medium. all procedures with live chikv were performed in a biosafety level facility at the leiden university medical center. a cdna clone of the synthetic chikv strain ls -gfp, which contains a duplicated subgenomic promoter and expresses the egfp reporter gene, was designed in silico as described in the results section. three dna fragments together forming a cdna copy of chikv ls -gfp were chemically synthesized (geneart, germany). using standard cloning techniques, these fragments were assembled and cloned into the asci-noti sites of vector puc an, a puc -derived plasmid in which the original polylinker was replaced by one with asci-ncoi-ecorv-xhoi-noti sites. the resulting plasmid (pchikv-ls -gfp) contains the genomic cdna of chikv ls -gfp directly downstream of a phi . promoter and followed by a unique spei linearization site for dna linearization prior to in vitro transcription. the 'wild type' synthetic pchikv-ls construct was made by deleting the bp egfp-containing saci fragment from pchikv-ls -gfp (fig. ) . a third variant with a duplicated subgenomic promoter and a multiple cloning site behind subgenomic promoter (pchikv-ls -mcs), which allows the introduction of e.g. a reporter gene, was generated by removing the bp asisi-paci fragment from pchikv-ls -gfp. the constructs were verified by sequencing. in vitro transcription and rna transfection rna was transcribed from plasmids with the phi . promoter [ ] using the ampliscribe t high yield transcription kit (epicenter), the m gpppa rna cap structure analog (neb) and . mg of template dna that had been linearized with spei. after a -h reaction at uc, template dna was digested with dnasei and rna was purified by precipitation with . m licl (ambion). the concentration of in vitro transcribed rna was determined with a nanodrop spectrophotometer (thermo scientific) and its integrity was checked by agarose gel electrophoresis. bhk- cells ( ) were electroporated with mg of rna using program t- of the amaxa nucleofector and kit t (lonza) according to the manufacturer's instructions. electroporated cells were plated in well clusters and incubated at uc in the same medium used for chikv infection experiments. chikv rna was isolated from virions using the qiaamp viral rna mini kit. four overlapping amplicons were generated by a two-step reverse transcriptase (rt) pcr. in the first step cdna was synthesized using revertaid h minus reverse transcriptase (fermentas) and primers at- (gactgca-gatgcccgccatt), at- (cgctcggtccagg-caactct), at- (cgtggtgtttgccaacaggc), or at- (cgccgttttttttttttttttttttttttt). in the second step pcr products were generated using combinations of primers at- (atggctgcgtgagacacacg) and at- , at- (tgcacccaagtgtaccacaa) and at- , at- (caggagagtgcatccatggc) and at- , or at- (gaatgcgcgcagatacccgt) and at- . the resulting rt-pcr products were purified and directly sequenced ( ng template) using the bigdye terminator cycle sequencing kit v . (applied biosystems) and a genetic analyzer automatic sequencer (applied biosystems). pcr conditions and primer sequences are available upon request. viral titers were determined by plaque assay on vero e cells. six-well clusters containing confluent monolayers of vero e cells were incubated with . -ml volumes of -fold serial dilutions of chikv-containing samples. after a -h incubation at uc, the inoculum was replaced with ml of dmem containing . % avicel rc- (fmc biopolymer), % fcs, mm hepes, and antibiotics. after a -h incubation at uc, monolayers were fixed with . % formaldehyde in pbs and plaques were visualized by crystal violet staining. for infectious center assays -fold serial dilutions of electroporated cells were added to -well clusters already containing a monolayer of bhk- cells per well. after a -h incubation at uc, a dmem/avicel overlay was applied and cells were incubated at uc for h. plaques were visualized as described above. total rna was isolated from cells by lysis in . ml of mm tris-hcl (ph . ), mm licl, mm edta, mm dtt, % (w/v) lithium dodecyl sulfate, and mg/ml proteinase k. after acid phenol (ambion) extraction, rna was precipitated with isopropanol, washed with % ethanol, and dissolved in mm sodium citrate (ph . ). samples containing rna from . cells were mixed with volumes of % formamide, % formaldehyde, . % glycerol, mm mops (ph . ), . mm naac, . mm edta, . % sds, and . % bromophenol blue. after denaturation for min at uc, rna was separated in . % denaturing formaldehyde-agarose gels using the mops buffer system as described [ ] . rna molecules were detected by direct hybridization of the dried gel with p-labeled oligonucleotides essentially as described previously [ ] . positive-stranded genomic and subgenomic chikv rnas were visualized with probe chikv-hyb ( -tgtgggttcggagaatcgtg-gaagagtt- ) that is complementary to the end of the genome. negative-stranded rna was detected with probe chikv-hyb ( -aacccatcatggatcctgtgtacgtg-ga- ) that is complementary to the end of the minus strand. s ribosomal rna (loading control) was detected with the oligonucleotide probe -atgcccccggccgtccctct- . probes ( pmol) were labeled with mci [c- p]atp (perki-nelmer) in a h reaction using u of t polynucleotide kinase (invitrogen) in ml of the supplied forward reaction buffer (invitrogen). prehybridization ( h) and hybridization (overnight) were done at uc in sspe ( . m nacl, mm nah po , mm edta, ph . ), denhardt's solution, . % sds, and . mg/ml homomix i. hybridized gels were washed twice in sspe with . % sds before they were exposed to storage phosphor screens. after scanning with a typhoon- scanner (ge healthcare), quantification of rna levels was done with quantity one v . . (biorad) and corrections for loading variations were made based on the quantity of s ribosomal rna in the same lane. the results of two or three independent experiments were quantified (one representative experiment is shown in figures). total protein samples were prepared by lysing cells in . ml of laemmli sample buffer ( mm tris-hcl, ph . , % glycerol, % sds, mm dtt, , mg/ml bromophenol blue). proteins were separated by sds-page in % polyacrylamide gels and were transferred to hybond-lfp membranes (ge healthcare) by semi-dry blotting. after blocking with % casein (sigma) in pbs with . % tween- (pbst), membranes were incubated overnight with rabbit antisera against chikv nsp (raised using the peptide eveprqvtpndhan), nsp (raised using the peptide assrsnfeklrgpv) or e [ ] in pbst with . % casein. mouse monoclonal antibodies against b-actin (sigma), or the transferrin receptor (zymed) were used for detection of loading controls. biotin-conjugated swine-a-rabbit (dako) or goat-a-mouse (dako), and cy -conjugated mousea-biotin (jackson) were used for fluorescent detection of the primary antibodies with a typhoon- scanner (ge healthcare). at various time points post infection approximately chikv-infected or mock-infected /ace cells in -well clusters were incubated with mci of h-uridine in medium and incorporation was allowed to proceed for minutes at uc. total rna was isolated and analyzed in a denaturing agarose gel as described above. for fluorographic detection of h-labeled rna, the gel was soaked in methanol for hour (one change) and then incubated with % , -diphenyloxazole in methanol for at least hours. after incubation in milliq for minutes, the gel was dried and a fuji rx film was placed on top. films were developed after a - day exposure at uc and scanned with a biorad gs- densitometer. to check equal sample loading, the gel was hybridized with a p-labeled s ribosomal rna-specific probe as described above. in addition, incorporation of h-uridine into rna was quantified by analyzing -ml samples of isolated total rna with a liquid scintillation counter (beckman ls ic). in control samples, cellular transcription was inhibited by adding actinomycin d (sigma) to a final concentration of mg/ml. at various time points post infection approximately chikv-infected or mock-infected /ace cells in -well clusters were starved in dmem lacking l-methionine and lcysteine (invitrogen) for min., and subsequently incubated with mci easytag express s protein labeling mix (perkinelmer) for min. total protein samples were analyzed by sds-page as described above. gels were stained with coomassie to check equal sample loading and s-labeled proteins were detected by drying the gels and exposing them to a storage phosphor screen, which was scanned - days later with a typhoon- scanner (ge healthcare). chikv-or mock-infected vero e cells grown on coverslips were fixed with % paraformaldehyde in pbs. after quenching with mm glycine in pbs, cells were permeabilized with . % triton in pbs for min. and coverslips were incubated with primary antibodies diluted in pbs with % fcs for h. doublestranded rna was detected with a : dilution of mouse monoclonal antibody j (english & scientific consulting). chikv e was visualized with a : dilution of a polyclonal rabbit antiserum [ ] . detection of primary antibodies was done with donkey-a-mouse-cy , goat-a-rabbit-cy or goat-a-rabbit-alexa ( : ; jackson). nuclei were stained with hoechst . the coverslips were mounted with prolong (invitrogen) and analyzed using an axioskop mot plus fluorescence microscope with axiocam hrc camera and axiovision software (zeiss). mouse monoclonal antibodies raised against chikv particles of strain ita -ra (izsler, brescia, italy) were heatinactivated for min. at uc. two-fold serial dilutions of the neutralizing monoclonal antibody h and non-neutralizing control antibody h [ ] were incubated with an equal volume of medium containing pfu of chikv. these mixtures were incubated for min. at uc and transferred to -well clusters containing vero e cells per well. after incubation at uc for days, the wells were fixed with . % formaldehyde and cpe was detected by staining with crystal violet. chloroquine, -aza-uridine and ribavirin were dissolved in pbs. cyclosporin a and -deaza-adenosine were dissolved in dmso. mycophenolic acid was dissolved in ethanol. all compounds were obtained from sigma. for cpe reduction assays, -well clusters with , vero e cells/well were incubated with pfu of virus per well, corresponding to a multiplicity of infection (moi) of . , and -fold serial dilutions of the compound in medium. wells without cells, uninfected cells, infected untreated cells and infected cells treated with solvent alone were included as controls. four days post-infection cell viability was assessed using the celltiter h aqueous non-radioactive cell proliferation assay (promega). cpe reduction experiments with ribavirin were done with bhk- cells in a similar way, except that viability was assessed days post infection. for egfp reporter gene assays, , vero e cells/well in black -well plates were infected with chikv ls -gfp at an moi of . . after a -h incubation in medium containing the compound, the cells were fixed with % paraformaldehyde in pbs. egfp expression was quantified using a berthold mithras lb plate reader, with excitation and emission wavelengths of and nm, respectively. the fluorescence in wells containing mock-infected cells was used to correct for background signal. ic and cc values were calculated with graphpad prism using the nonlinear regression model. all animal experiments described in this paper were carried out in the bsl facilities of the erasmus medical center in accordance with the dutch guidelines for animal experimentation and were approved by the institute's independent animal ethics committee. twelve-day old c bl/ mice were injected intraperitoneally with tcid of chikv s , chikv ls or chikv ls -gfp. after the challenge the mice were monitored daily for signs of illness or death. the infection was considered lethal when the animals reached humane end-points and needed to be euthanized. viral rna was extracted from brain samples using the automated magnapure method (total nucleic acid isolation kit, roche diagnostics, the netherlands) according to the manufacturer's instructions, and quantified using a one-step rt-pcr taqman protocol (ez-kit, applied biosystems) and an abi prism detection instrument. the primers and probe used for chikv rna quantification were essentially as described [ ] except that probe fam-ccaatgtcttcagcctgga-caccttt-tamra was used. dilutions of virus suspensions of known titer were included to make a calibration curve, which was used to express results as tcid equivalents per gram of brain tissue. all animal experiments described in this paper were carried out in the bsl facilities of the erasmus medical center in accordance with the dutch guidelines for animal experimentation. a dutch government-approved and independent animal experimentation ethical review committee (stichting dec consult) approved the animal studies (permit nr. emc / - - ). the genbank accession numbers for the full-length cdna clones pchikv-ls , pchikv-ls -gfp and pchikv-ls -mcs are jx , jx , and jx respectively. the genbank accession numbers for the genomic rna sequences of chikv ls , ls -gfp, lcs -mcs and nl / are kc , kc , kc , and kc , respectively. the complete genomes of the chikv strains carrying the e -a v mutation ( table ) that were available in genbank at the time of in silico design (november ) were aligned using mafft [ ] and the resulting consensus sequence formed the basis for the synthetic full-length cdna clones. a nucleotide polya tail was added to the end of the consensus sequence and an a g point mutation was introduced to create a translationally silent saci restriction site required for cloning. the virus encoded by the resulting sequence was termed chikv ls (genbank accession kc ). variants containing a duplicated subgenomic promoter and a multiple cloning site (chikv ls -mcs; genbank kc ) or an egfp reporter gene (chikv ls -gfp; genbank kc ) were also designed. the egfp reporter gene was placed under control of the native subgenomic promoter and upstream of a second subgenomic promoter that drives expression of the viral structural polyprotein, as this configuration was previously reported to result in a more stable reporter gene expression [ ] . the chikv cdnas were placed downstream of a phi . t promoter, and a unique spei site for linearization prior to in vitro transcription directly followed the polya tail. the phi . promoter was used because the ends of capped transcripts generated from this promoter with t polymerase and the m gpppa cap analog are identical to the end of the genomes of naturally occurring chikv strains. in contrast, capped rnas generated by in vitro transcription from the frequently used sp promoter will contain m gpppg at their terminus, i.e. will contain an additional terminal g residue. however, existing chikv cdna clones that contain the sp promoter also efficiently yield infectious virus and it is assumed that the additional -terminal g residue is removed during subsequent rounds of replication. in line with this, in vitro transcribed rna from pchikv-ls , a variant of plasmid pchikv-ls in which the phi . promoter was replaced with the sp promoter also yielded infectious virus. plasmid pchikv-ls -gfp, the infectious clone encoding the egfp-expressing reporter virus, was created by assembling the chemically synthesized dna fragments as described in the materials and methods section. plasmid pchikv-ls , the infectious clone encoding the synthetic 'wild type' strain chikv ls , and plasmid pchikv-ls -mcs were generated from pchikv-ls -gfp by deleting specific restriction fragments, as described in materials and methods (fig. ) . in the original alignment, strains drde- (genbank u ) and d / (genbank ef ) shared the highest sequence similarity with ls , with amino acid differences (table ) . however, a blast search performed in march , three years after the design of chikv ls , and alignment of the retrieved complete chikv genomes revealed that strains ind- -ap (genbank ef ), ind-gj (genbank fj ), and chik (genbank eu ) share the highest degree of nucleotide sequence identity with chikv ls (. . %), with only - nucleotide differences respectively (table s ). interestingly, these indian strains were not included in the original alignment on which the ls sequence was based, as they do not contain the e -a v mutation (table ) . however, nsp of ls is identical to that of ind- -ap . at the amino acid level, chikv ls differs at positions from lr _opy and at positions from ita -ra (table ). to determine whether infectious virus could be generated from the synthetic chikv clones, in vitro transcribed rna was electroporated into bhk- cells. strong egfp fluorescence was readily detected h after transfection of chikv ls -gfp rna. for chikv ls and ls -gfp rna specific infectivities of approximately pfu/mg of rna were found in infectious center assays, which is similar to what has been found for other chikv cdna clones [ , ] . virus titers in cell culture supernatants h after electroporation, were generally in the range of - pfu/ml. this is lower than the peak viral titers that are obtained during infection experiments, but can be explained by the early time point of harvesting and the fact that not all cells were transfected. as expected, electroporation of bhk- cells with uncapped chikv rna did not result in the release of infectious virus. to assess the stability of egfp reporter expression, chikv ls -gfp was serially passaged (moi . ) in both /ace and vero e cells. virus harvested during each passage was used to infect vero e cells at an moi of . and immunofluorescence microscopy revealed that at passage , over % of the e positive foci still displayed robust egfp expression. sequencing of cdna obtained by rt-pcr amplification of rna extracted from extracellular virions revealed that, after passages on vero e cells, the consensus genome sequence of chikv ls -gfp was identical to the original in silico designed sequence. these results demonstrated that the synthetic viruses are viable, genetically stable, and able to retain stable expression of the reporter gene. since we aim to use chikv in sirna screens and proteomics studies to identify host factors involved in replication, various human cell lines were evaluated for their ability to support chikv replication. chikv ls -gfp was able to productively infect hela, mrc- , huh , , and /ace cells (data not shown). infection of hela and huh cells was not very efficient and these cells were therefore not used for any further experiments. /ace cells were selected for this study, as they supported high levels of chikv ls -gfp replication, could be efficiently transfected with sirnas, and -unlike regular cellsadhered well to tissue culture plastics. /ace cells stably express angiotensin-converting enzyme (ace ), the receptor for sars-coronavirus. ace expression is not required for chikv infection, but these cells were chosen because of the aforementioned advantages and the fact that they have been previously used in our lab in sirna screens for host factors that affect corona-and arterivirus replication ( [ ] ; de wilde et al. submitted; wannee et al., in preparation). using these cells in similar sirna screens with chikv and other alphaviruses would allow direct comparison of data sets with those obtained for corona-and arteriviruses, which could lead to the identification of common (broad spectrum) pro-and antiviral host factors. to determine whether the synthetic viruses behave like natural isolates, their growth kinetics in vero e , /ace , and c / cells were compared to those of ita -ra , which was isolated during the chikv outbreak in italy ( fig. a-c) . the growth curves of chikv ls on all three cell lines were found not to differ significantly from those of ita -ra , with virus titers reaching a maximum - h post infection (p.i.). peak virus titers on mosquito cells were approximately -log higher than those on mammalian cells. chikv ls -gfp replicated slightly slower than the other viruses in all three tested cell lines, which is not unusual for recombinant reporter viruses. egfp expression could be detected as early as h p.i. and peaked around h p.i. the plaque morphology of the synthetic viruses was similar to that of ita -ra (fig. d) . chikv ls induced a cytopathic effect (cpe) indistinguishable from the natural isolate. on vero e cells early signs of cpe started to appear around h p.i. and cpe was complete by h p.i. (fig. e) . to study chikv-induced transcriptional host shut-off, the incorporation of h-uridine into cellular and viral rna was analyzed by metabolic labeling of infected /ace cells at various time points post infection (moi of ). a strong reduction in the incorporation of h-uridine into rna was observed at - h p.i. in cells infected with chikv ls or ita , as determined by liquid scintillation counting of total rna samples table . comparison of the amino acid sequences of chikv ls and various natural isolates. the chikv ls amino acid sequences were aligned with those of several highly similar natural isolates, and clinical isolate nl / . only amino acid differences are indicated and identity is represented by dots. the numbering is based on the sequence of ls , which is also equal to that of lr _opy . it is important to note that -like all other chikv strains in this ( fig. a) . inhibition of cellular transcription with actinomycin d for min. prior to metabolic labeling at h p.i. revealed the contribution of viral rna synthesis to the total signal. fluorographic detection of h-labeled rna analyzed in denaturing gels also showed a decrease in cellular transcription during the course of the infection, while the synthesis of chikv rna became clearly detectable by h p.i (fig. b) . transcriptional shut-off occurred around - h p.i. and was induced by chikv ls and ita with similar kinetics, although ls seemed to act slightly faster. to examine chikv-induced translational shut-off, the synthesis of s-labeled viral and cellular proteins during the course of chikv ls infection was analyzed by metabolic labeling of infected /ace cells with s-met and s-cys (fig. c) . a clear shut-off of host translation was observed - h p.i. beyond h p.i. the bulk of newly produced protein appears to be of viral origin, likely c, e , e and their precursors (indicated with * in fig. c ). chikv ita and ls induced translational host shut-off in a similar manner (only results obtained with ls are shown in fig. c ). both chikv ita -ra and the synthetic viruses established non-cytopathic persistent infections in c / mosquito cells. all characterization experiments have been performed in both / ace and vero e cells, with similar results. for simplicity only the results for /ace cells are shown, except for immunofluorescence experiments, which were done with vero e cells as they had a more suitable morphology. the replication cycle of the synthetic viruses and ita -ra was characterized in more detail to assess whether the synthetic viruses behaved like their natural counterpart. the kinetics of rna synthesis was analyzed by isolating total rna from / ace cells infected with chikv ls , ls -gfp, or ita -ra at various time points post infection. negative-and positivestranded rnas were detected by hybridization with p-labeled oligonucleotide probes (fig. a ). both negative-and positivestrand rnas were readily detected in cells infected with the various strains starting at h p.i. the negative-strand rna was less abundant than the positive strand, it was easily detected relatively early in infection ( fig. a top panel, fig. b) , and appeared to decrease at later time points as has also been observed for other alphaviruses. this apparent decrease is probably not only due to degradation of minus strands, but at least partly due to a hampered detection caused by the large excess of positive-strand rna present at late time points. this excess of positive-strand rna competes with the radioactively labeled minus-strand specific probe. in support of this, we observed that mixing rna isolated from chikv-infected cells at h p.i. with in vitro transcribed positive-strand rna reduced the amount of negative strand that could be detected (data not shown). furthermore, when samples taken at and h p.i. were treated with singlestrand-specific rnase a/t before hybridization, the negativestrand levels at the late time point were approximately % of that at h p.i, instead of the approximately % in untreated samples (data not shown). using a positive-strand-specific probe, both the s genomic and s sgrna could be detected, and both rnas accumulated until h p.i (fig. a middle panel, fig. c ). the ratio of genomic to sgrna varied between : . and : . during the course of infection, similar to the ratios reported for semliki forest virus and sinv [ ] . the kinetics of rna synthesis and rna accumulation levels were similar in chikv ls -and ita -ra -infected cells. in cells infected with chikv ls -gfp, the additional subgenomic rna encoding the egfp reporter gave rise to an extra band above the s rna band, and its expression level was calculated to be approximately half of that of the s rna. the individual levels of the two sgrnas expressed by chikv ls -gfp were lower than those of ita or ls , but their combined abundance was comparable to that of the viruses expressing a single sgrna (fig. c ). to monitor viral protein expression, /ace cells were infected with chikv ls , ls -gfp, or ita -ra and total protein was isolated at various time points post infection. these samples were analyzed by western blotting with antisera against the nonstructural proteins nsp and nsp , and the structural protein e . expression of nsp , e , and the e e precursor could be detected as early as h p.i. and the proteins accumulated over time, reaching a plateau around h p.i. (fig. ) . the rdrp nsp could not be detected in infected cells using a chikv nsp specific antiserum capable of detecting the purified bacterially expressed protein. this was probably due to the low affinity of the antibody, the low expression level and relative instability of nsp in infected cells, as was also observed for other alphaviruses [ ] . in addition, a quantitative proteomics study on chikv-infected cells also suggested that at h p.i. the amount of nsp was at least -fold lower than that of nsp (treffers, tas, de ru, van veelen, snijder and van hemert, manuscript in preparation). indirect immunofluorescence analysis of vero e cells infected with chikv ls , ls -gfp, or ita -ra at various time points showed that the localization and expression kinetics of e and dsrna were similar for the natural isolate and the synthetic viruses (fig. ) . double-stranded rna, which is assumed to be generated during replication of chikv in infected cells [ ] , could be detected as early as h p.i. and remained clearly visible throughout the infection. the dsrna localized to foci throughout the cytoplasm. the e protein could be detected from h p.i. onwards with maximum expression reached by h p.i. the e protein mainly localized to the plasma membrane of infected cells. egfp produced by the reporter virus was visible from h p.i. onwards, reaching a maximum level around h p.i. (fig. c ). chikv ls and ita -ra were compared in a neutralization assay using the neutralizing monoclonal antibody h that was raised in mice against chikv ita -ra virions, and appears to recognize a linear epitope in e [ ] . the nonneutralizing mab h was used as a control. both the natural isolate and chikv ls were neutralized with similar characteristics by h , while their infectivity was not affected by h (fig. ) . newborn mice are highly susceptible to chikv infection and they develop symptoms as lethargy, dragging of hind limbs, flaccid paralysis, and reduced weight gain [ ] . -day old mice were injected intraperitoneally with tcid of chikv ls , ls -gfp or prototype strain s as a control. the animals were euthanized when their humane end points were reached or days post infection and viral rna levels in brain tissue were analyzed (fig. ) . both synthetic viruses behaved like the natural isolate in vivo, causing lethal infections with similar kinetics (fig. a ). in addition, the viral titers in the brains of chikv s -infected mice were similar to those of mice infected with the synthetic viruses (fig. b ). to evaluate their suitability for analyzing the potency and mechanism of action of antiviral compounds, the sensitivity of chikv ls and ls -gfp to a number of such compounds was determined and compared to ita -ra . cyclosporin a, which through its effect on cellular cyclophilins inhibits the replication of a variety of viruses, had no specific effect on chikv replication, not even at a (cytotoxic) dose of mm (data not shown). the compounds -deaza-adenosine, -aza-uridine, chloroquine, and mycophenolic acid were tested in cpe reduction assays with vero e cells infected at an moi of . and analyzed days p.i. they were all found to inhibit chikv replication with ic s in the low micromolar range and with minimal cytotoxicity (fig. a-d) . no substantial differences were observed between the ic values calculated for ita -ra , ls and ls -gfp. the four compounds also clearly reduced egfp reporter gene expression in vero e cells infected with chikv ls -gfp (fig. f) . slightly lower ic values were obtained for -aza-uridine and chloroquine, and a significantly higher ic was observed for -deazaadenosine in this assay, compared to the cpe-based assay. this might be due to the mode of action of -deaza-adenosine and/or due to differences in experimental set-up compared to the cpebased assay (moi . vs. . ; measurement h p.i. vs. d p.i). ribavirin is a known inhibitor of chikv replication, but in our cpe reduction assay with vero e cells it was not very effective in inhibiting replication of the various strains, as ic values of over mm were obtained (fig. e, gray lines) . this is likely due to the inefficient conversion of ribavirin to its active phosphorylated form in vero e cells [ ] . therefore, we have also analyzed the effect of ribavirin in a -day cpe reduction assays with bhk- cells, which are able to metabolize ribavirin [ , ] and found ic s of - mm for the various strains. clinical isolate nl / was also included in the assays and appeared to be somewhat more sensitive to the antiviral compounds than ls and ita -ra . however, the slower replication kinetics of this strain made it impossible to directly compare nl / and ls in the same cpe reduction assays, despite the fact that virus yields and cytopathic effect of nl / and ls were comparable (data not shown). the massive chikv outbreaks that have been occurring in asia and the indian ocean region since are associated with the emergence of strains with the a v substitution in the e glycoprotein, which allowed their transmission by a novel mosquito vector, aedes albopictus [ ] [ ] [ ] [ ] [ ] [ ] . these east-central-south african lineage-derived strains even appear to be replacing the the relative abundance of rna was calculated as before, except that data were normalized to the value measured for ls sgrna at h p.i ( %). genomic rna levels are indicated with black lines, sgrna levels with gray lines. the total level of both sgrnas expressed by ls -gfp is indicated with the gray dotted line. doi: . /journal.pone. .g asian lineage chikv strains that have been endemic in the region for decades. since the s, the geographic distribution of aedes albopictus has dramatically expanded and now also includes large parts of the usa and several european countries. this creates concern for locally transmitted outbreaks in europe and the usa, which could be initiated by viraemic travelers arriving from countries where chikv is endemic, like india and indonesia. locally transmitted chikv infections have indeed already been reported from italy in and france in [ , ] and recent studies suggest that also the usa is at risk for locally transmitted chikv outbreaks [ , ] . besides its large medical and societal impact in endemic countries, the increased risk of chikv outbreaks in europe and the usa underlines the importance of studying the replication of this important human pathogen and its interactions with the host to develop safe and effective vaccines and antiviral therapy. infectious cdna clones have proven to be important tools to study many aspects of the viral life cycle, and molecular clones of a variety of natural isolates have been instrumental in several recent chikv studies [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the existing chikv molecular clones can be considered to be derived from a single genome (or fragments of single genomes) out of the whole spectrum of viruses present in a chikv quasispecies population. in contrast, most of the complete chikv genome sequences that have been deposited in genbank represent the consensus (or master sequence) of a viral quasispecies population. the diversity (and evolution) of a chikv quasispecies population has probably been shaped by the characteristics of the individual host and the specific tissue source (serum) from which it was isolated. for ross river virus it was observed that the level of intrahost genetic variation in patient serum samples, was considerably larger than that observed at the epidemiological scale, which can be explained by the purifying selection for replication in both arthropod and vertebrate hosts [ ] . advances in sequencing techniques now allow a more detailed view on quasispecies diversity and intrahost evolution, and also for chikv a recent study has provided more insight into quasispecies dynamics and the effect of purifying selection by host alternation [ ] . a link was observed between increased fitness as a result of alternating passaging and reduced quasispecies complexity, which restricted adaptability to novel selective pressures like antiviral treatment or antibody-mediated neutralization [ ] . individual chikv isolates or molecular clone derived viruses could have their specific properties in terms of replication kinetics, vector specificity, dissemination within the host, virulence, virus-host interactions or sensitivity to antiviral compounds. we were interested in studying the general characteristics of the life cycle and virus-host interactions of the e - v chikv strains that were circulating during the - outbreaks. therefore, we have constructed a fully synthetic cdna clone, chikv ls , based on the consensus sequence of the aligned genomes of these recent e - v isolates, rather than on a single genome from a clinical isolate. in addition, a variant that expresses the egfp reporter gene under control of a (duplicated) subgenomic promoter was created (chikv ls -gfp). the current possibilities of gene synthesis allowed the design of these clones in silico, with sequences tailored to our requirements, e.g. already containing a reporter gene under control of a duplicated subgenomic promoter and including (translationally silent) mutations to create restriction sites that facilitate cloning and reverse genetics studies. alignment of all complete chikv genomes that were in genbank by june yielded a consensus sequence that differed only at nucleotide positions from the sequence of ls that was designed years earlier. these were position , at which we introduced a translationally silent restriction site (g a), a synonymous urc substitution at position , , and position , , which is a c in % of all deposited genomes (strains with e - a), while the remaining (e - v) strains have a u at this position. an interesting observation was that % of the sequenced chikv strains, including the prototype strains s and ross, contain an arginine codon instead of the opal stop codon that is present between the nsp -and nsp -coding regions of most chikv isolates. the presence or absence of this opal codon might be influenced by the passage history of the isolate as has been observed for other alphaviruses [ ] . this might also explain why the sequence of the original clinical isolate of lr -opy (genbank dq . ) contains the opal termination codon near the end of the nsp coding region, while the infectious clone of this strain (genbank eu . ) contains an arginine codon at this position. to assess whether the synthetic viruses are representative models, their characteristics were compared to those of the natural strain ita -ra . like the natural isolate, the synthetic viruses caused cytopathic infections in vero e and /ace cells (fig. e) , whereas non-cytopathic persistent infections were observed in the mosquito cell line c / . in vertebrate cells all strains caused a shut-off of cellular translation around - h p.i. and a strong inhibition of cellular transcription by - h p.i. the accumulation of negative-and positive-strand viral rna, the kinetics of non-structural and structural viral protein expression, as well as the growth kinetics and plaque morphology of the synthetic viruses were indistinguishable from those of chikv ita -ra ( fig. - ). in addition, the synthetic viruses caused lethal infections in -day old mice, with virus spreading to the brain, as observed for natural isolates (fig. ) . although this demonstrates that the synthetic viruses replicate in vivo, this mouse model does not allow comparison of strains for more subtle differences in virulence and pathogenesis. the genomic stability of chikv ls -gfp was assessed and after passages its (consensus) sequence was found to be identical to the original in silico designed sequence. the expression of the egfp reporter gene was stable for at least passages, making the synthetic viruses suitable tools for highthroughput screens for antiviral compounds, (reverse genetics) studies into their mechanism of action, and systematic functional genomics screens for host factors affecting chikv replication. to evaluate whether chikv ls and ls -gfp are suitable to analyze the potency and mechanism of action of antiviral compounds, their sensitivity to a number of such compounds was determined and compared to ita -ra . the lysosomotropic agent chloroquine and nucleoside analog -aza-uridine inhibited the replication of the synthetic viruses and natural isolates with ic s that were in the same range and comparable to values previously reported by others [ ] [ ] [ ] [ ] [ ] [ ] . the inhibitory effect of chloroquine on the replication of many viruses including alphaviruses has been known for decades. for chikv it is a useful reference compound in cell-based studies, but a small scale clinical trial on the island of la reunion suggested it is not effective in the treatment of chikv infections in patients [ ] . the nucleoside analog -aza-uridine has previously been reported to inhibit the replication of a variety of viruses, including chikv [ , ] . the compound could interfere with cellular utp metabolism and may be incorporated into chikv rna, leading to chain termination and/or increased error frequency, ultimately resulting in 'error catastrophe'. mycophenolic acid is a noncompetitive inhibitor of inosine monophosphate dehydrogenase (impdh), causing a depletion of the intracellular guanosine pool. it is a known inhibitor of various viruses, including chikv [ , ] . ribavirin is a synthetic nucleoside analog with broad spectrum antiviral effect due to potential effects on the cellular impdh enzyme, viral rna synthesis and capping [ ] . however, not all cell lines are able to perform the necessary conversion of this compound to its active phosphorylated form, explaining the contradictory reports on the antiviral activity of this compound [ , , ] . in our hands, ribavirin inhibited chikv replication in bhk- cells with an ic of around mm, while it was hardly effective in vero e cells, with ic values of over m. the ic that we obtained with bhk- cells is in the same range as those previously reported for the antiviral effect of ribavirin on chikv replication [ , ] . cyclosporin a, which through its effect on the cellular cyclophilins, inhibits the replication of a variety of viruses (for recent review see [ ] ), had no effect on chikv replication. we identified -deaza-adenosine as a novel inhibitor of chikv replication with an ic of approximately mm and a cc . mm. this compound has previously been identified as inhibitor of a broad spectrum of viruses, although many other +rna viruses appeared to be rather insensitive or not affected at all (reviewed in [ ] ). the antiviral activity of -deazaadenosine was attributed to its inhibitory effect on the cellular enzyme s-adenosylhomocysteine hydrolase, leading to an accumulation of s-adenosylhomocysteine, which inhibits s-adenosylmethionine-dependent methylation reactions [ ] . in this manner the enzyme plays a key role in s-adenosylmethionine-dependent methylation reactions and inhibition of viral methylation reactions (e.g. of viral rna) apparently can be achieved at compound concentrations that do not notably interfere with cellular methylation reactions. our observation warrants a more detailed analysis of the mode of action of -deaza-adenosine and analogs, also to evaluate their potential for use in antiviral therapy to treat chikv infections. overall, no large differences were observed between the ic values calculated for ita -ra , ls and ls -gfp, indicating that the synthetic viruses are suitable for use in antiviral screens. for most compounds, a faster and simpler assay with chikv ls -gfp reporter virus showed a good dosedependent response that correlated well with results obtained in the cpe-based assay. clinical isolate nl / exhibited slightly slower replication kinetics and appeared to be more sensitive to antiviral compounds than ita and the synthetic viruses. differences in the sensitivity to antiviral compounds among clinical isolates is not an uncommon phenomenon. nl / differs at amino acid positions from ls and it will be interesting to determine the contribution of these mutations, in particular the r s substitution in nsp , to the slower replication kinetics (and higher sensitivity to antivirals). taken together the detailed characterization of the chikv replication cycle at the molecular level demonstrated that our new synthetic consensus-based viruses behave like natural isolates and are suitable tools to study various aspects of the chikv life cycle, which should ultimately provide a basis for the development of antiviral therapy. table s comparison of chikv ls with the genome sequences of various closely related natural isolates. only differences between ls and each of the other strains are summarized. dots indicate that the nucleotide at that position is identical to that at the corresponding position in the sequence of ls . genomes were aligned with mafft and analyzed in jalview. numbering is based on the sequence of lr _opy (and is equal to ls numbering). the nucleotide at position (indicated in gray) determines whether the strain has the a v mutation in the e protein. strains with a t at this position have the a v mutation. differences not included in the comparison are the nt, nt and nucleotides that are missing from the 'utr of the sequences of drde- , d / and ita -ra , respectively. the missing first nt, missing last nt and the insertion of an a after position in the sequence of ind- -ap were also not included in this comparison. 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of vesicular stomatitis virus and sendai virus to ribavirin cyclophilin involvement in the replication of hepatitis c virus and other viruses carbocyclic adenosine analogues as s-adenosylhomocysteine hydrolase inhibitors and antiviral agents: recent advances we are grateful to dr. gorben pijlman (wageningen university, the netherlands) for his generous gift of chikv e antiserum and to adriaan de wilde and emmely treffers for helpful discussions and sharing their unpublished data. key: cord- -a oejky authors: sasaki, michihito; uemura, kentaro; sato, akihiko; toba, shinsuke; sanaki, takao; maenaka, katsumi; hall, william w.; orba, yasuko; sawa, hirofumi title: sars-cov- variants with mutations at the s /s cleavage site are generated in vitro during propagation in tmprss -deficient cells date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: a oejky the spike (s) protein of severe acute respiratory syndrome-coronavirus- (sars-cov- ) binds to a host cell receptor which facilitates viral entry. a polybasic motif detected at the cleavage site of the s protein has been shown to broaden the cell tropism and transmissibility of the virus. here we examine the properties of sars-cov- variants with mutations at the s protein cleavage site that undergo inefficient proteolytic cleavage. virus variants with s gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. these alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type ii transmembrane serine protease, tmprss . notably, viruses with s gene mutations emerged rapidly and became the dominant sars-cov- variants in tmprss -deficient cells including vero cells. our study demonstrated that the s protein polybasic cleavage motif is a critical factor underlying sars-cov- entry and cell tropism. as such, researchers should be alert to the possibility of de novo s gene mutations emerging in tissue-culture propagated virus strains. was the dominant pathway employed by sars-cov- in tmprss -expressing cells [ ] . in contrast, camostat had no impact on entry of the s gene mutant, del , into vero-tmprss cells but e- d treatment resulted in a dose-dependent decrease in del entry (fig. a) . these results suggested that s gene mutant, del , can enter vero-tmprss cells via cathepsin-dependent endocytosis but not the tmprss -mediated fusion pathway. parental vero cells that do not express tmprss were inoculated with s gene mutant viruses in the presence of camostat and/or e- d. addition of e- d inhibited the entry of all s gene mutants into both vero-tmprss and parent vero cells; by contrast, camostat had no impact on s gene mutant entry into these target cells (fig. b ). these results suggested that, in contrast to wt virus, s gene mutants enter into cells via cathepsin-dependent endocytosis only, regardless of the presence or absence of tmprss . because wt virus and s gene mutants showed different sensitivities to the treatment with camostat, an agent currently under exploration as a candidate antiviral for clinical use [ ], we also examined the impact of other antiviral agents including nafamostat (a tmprss inhibitor) [ , ] and remdesivir (a nucleotide analog) [ , ] . antiviral effects in vero-tmprss cells were estimated by a cell viability assay based on the generation of cytopathic effects [ ] . consistent with previous studies [ , ] , nafamostat showed higher antiviral efficacy against wt virus than was observed in response to camostat; however, nafamostat had no antiviral activity against the s gene mutants ( table ). in contrast, remdesivir inhibited infection of both wt and s gene mutants with similar ec values (table ). these results indicated that s gene mutants are resistant to the treatment with tmprrss inhibitors, but are sensitive to antivirals that target post entry in an effort to understand the selection mechanisms underlying the generation of these mutant variants, we estimated the frequency of s gene mutants in virus population of sars-cov- that had undergone serial passage in cultured cells. sars-cov- from an original virus stock was underwent passage once (p ) to four times (p ) in vero or up to eight times (p ) in vero-tmprss . nucleotide sequence heterogeneity at the s /s cleavage site was determined by deep sequencing and variant call analysis. more than one million sequence reads from each passaged sample were mapped onto the s /s cleavage site and analyzed for sequence variation. no sequence variants were observed in virus populations until p in vero-tmprss (fig. a) . in contrast, nucleotide sequence deletions around the s /s cleavage site corresponding to del and del mutants were observed in all three biological replicates of sars-cov- populations passaged in vero cells (fig. a) . notably, wt nucleotide sequences were detected in fewer than % of the isolates evaluated at p and the wt was completely replaced with s gene mutants at p in vero cells. these results indicated that sars-cov- propagation in vero cells results in a profound selection favoring the s gene mutants. s gene mutants del and r h were not identified in the virus populations from p to p . an additional variant del with a deletion of amino acids at a point immediately upstream of the rrar motif (figs. s a and s b), was detected as a minor variant in sample # at p . these results suggest that these specific mutations occur only at low frequency. we then determined the frequency of s gene mutants in virus populations passaged in calu- and caco- , which are cells that endogenously express tmprss [ , ] , and also in t-ace that do not express tmprss [ ] . no s gene mutants were identified in sars-cov- passaged in calu- and caco- until p ; by contrast, s gene mutants emerged at p in t-ace cells (fig. b) . we also identified an additional variant r p carrying a single amino acid substitution at the rrar motif (figs. s a and s b) at p and p in t-ace cells (fig. b) . taken together, these results suggest a strong association between tmprss deficiency and the emergence of s gene mutants. trypsin is a serine protease that is typically added to culture medium to induce cleavage and activation of viral proteins, including the hemagglutinin (ha) protein of influenza virus and the fusion (f) protein of paramyxovirus to promote growth in tmprss -deficient cells [ ] . recent studies report that trypsin treatment activates sars-cov- s protein and induces syncytia formation in cells that transiently express the virus s protein [ , ] . as such, we examined whether exogenously added trypsin could compensate for tmprss deficiency and thus inhibit the emergence of s gene mutants during sars-cov- propagation in vero cells. deep sequencing analysis revealed that s gene mutants did emerge and accounted for the majority of the virus population after p in vero cells cultured in serum-free medium with added trypsin ( in this study, we isolated s gene mutants from sars-cov- wk- , a strain isolated difficult to impossible to maintain sars-cov- with the s /s cleavage site in its intact form. the present study characterized s gene mutants as sars-cov- variants that generate small plaques and that have a narrow range of cell tropism. the phenotypic alterations of s gene mutants might be explained by noting that the s gene mutants were unable enter target cells via direct fusion mediated by tmprss . indeed, a previous study demonstrated that the polybasic cleavage motif at the s /s cleavage site was indispensable for the entry of vsv-pseudotyped viruses into calu- cells that expressed tmprss [ ] . further studies using infectious s gene mutants will provide new insights into the role of the polybasic amino acid motif at the s /s cleavage site with respect to both sars-cov- infection and its pathogenicity. at this time, many studies are conducted using sars-cov- propagated in vero cells. considering the very real possibility that these virus stocks will accumulate s gene mutations, researchers must pay careful attention to the passage history of any working stocks of sars-cov- . moreover, we must be very objective when interpreting the results from studies using vero-passaged virus, especially those focused on s protein cleavage, cells were infected with either wt or s mutants of sars-cov- at an moi of . after h, cells were fixed with . % buffered formaldehyde, permeabilized with ice-cold methanol, and incubated with anti-sars-cov- s antibody (gtx , genetex). cells were inoculated with either wt or s mutants of sars-cov- at an moi of . . after h of incubation, cells were washed twice with phosphate-buffered saline (pbs) and cultured in fresh medium with % fbs. the culture supernatants were harvested at , , and h after inoculation. virus titers were evaluated by plaque assay. vero-tmprss were infected with either wt or s mutants of sars-cov- at - tcid and added to the plates. plates were incubated at for days, and cpe was determined for calculation of % endpoints using mtt assay. the concentration achieving % inhibition of cell viability (effective concentration; ec ) was calculated. the original stock of sars-cov- strain wk- was serially passaged in vero, vero-tmprss , calu- , caco- , and t-ace cells in complete culture medium or (for vero) in serum free dmem supplemented with . μ g/ml trypsin (gibco); three full-length s protein is cleaved into s and s proteins at the s /s cleavage site. functional domains (rbd, receptor binding domain; rbm, receptor binding motif) are highlighted. (b) multiple amino acid sequence alignments focused on the s /s cleavage site of wild type (wt) and isolated mutant viruses (del , del , del and r h). amino acid substitutions and deletions are shown as gray boxes, and the polybasic cleavage motif (rarr) at the s /s cleavage site is highlighted in red. a red arrowhead indicates s /s cleavage site. (a) vero-tmprss cells were infected with sars-cov- wt or del mutant in the presence of varying concentrations of the tmprss inhibitor, camostat, or the cathepsin b/l inhibitor, e- d, for h. at h post-inoculation, the relative levels of viral n protein rna were evaluated quantitatively by qrt-pcr. (b) vero-tmprss and vero cells were infected with sars-cov- wt or s gene mutants in the presence of μm camostat and/or μm e- d for h. at h post-inoculation, the relative levels of viral n protein rna were quantified by qrt-pcr. cellular β-actin mrna levels were used as reference controls. the values shown are mean sd of triplicate samples. one-way analysis of variance with dunnett's test was used to determine the statistical significance between the responses to treatment with inhibitors and the no-treatment controls; *p < . , **p < . , ***p < . . multiple (a) nucleotide and (b) amino acid sequence alignments were constructed based on the sequence of wt and sars-cov- variants identified by deep-sequencing (related to fig. ) . infectious viruses of del and r p were not isolated in this study. nucleotide substitutions and deletions are shown as gray boxes. sequence encoding the polybasic cleavage motif (rarr) at the s /s cleavage site is highlighted in red. sars-cov- was serially passaged in vero cells in serum free dmem containing trypsin with three biological replicates. nucleotide sequence diversity at viral s /s cleavage site was determined by deepsequencing. sars-cov- (r h) atcagactcagactaattctcctcggcgggcacatagtgtagctagtcaatccatcattgc multiple nucleotide sequence alignment of s /s cleavage site of wild type and isolated sars-cov- mutants nucleotide substitutions and deletions are shown as gray boxes. sequence encoding the polybasic cleavage motif (rarr) at the s /s cleavage site is highlighted in red sars-cov- (r p) atcagactcagactaattctcctccgcgggcacgtagtgtagctagtcaatccatcattgc sars-cov- (r p) ecdipigagicasyqtqtnspprarsvasqsiiaytmslgaensvaysnns ecdipigagicasyqtqt----------sqsiiaytmslgaensvaysnns sars-cov- (del ) ecdipigagicasyqtqtnspr-------qsiiaytmslgaensvaysnns sars-cov- (del ) ecdipigagicasyqtqtnsprrarsva---iiaytmslgaensvaysnns sars-cov- (r h) ecdipigagicasyqtqtnsprrahsvasqsiiaytmslgaensvaysnns key: cord- - ns onkw authors: kusters, inca c.; matthews, james; saluzzo, jean françois title: manufacturing vaccines for an emerging viral infection–specific issues associated with the development of a prototype sars vaccine date: - - journal: vaccines for biodefense and emerging and neglected diseases doi: . /b - - - - . - sha: doc_id: cord_uid: ns onkw abstract the world was struck by surprise when a severe acute respiratory syndrome (sars) epidemic started in in china. this disease had never been observed in man before; the sars-coronavirus causing the disease was unknown. with the uncertainty about the future impact of this epidemic, an important international collaboration started spontaneously sharing scientific knowledge and reagents. resources became quickly available, and public and private efforts were undertaken to develop rapidly a vaccine. we will discuss here the importance of the international collaboration and the availability of funding. moreover, we will review the most important and challenging steps during the industrial development of the sars vaccine highlighting the difficulties in terms of safety working with such a highly pathogenic, unknown virus. we will emphasize the industrial perspectives on inactivation and decontamination experiments, the selection of the most promising vaccine candidate, the production process and the choice and use of animal models in such a pressing and difficult situation. finally, we will briefly review the unique regulatory environment created during this period for the development of a sars vaccine. with the uncertainty about the possible extent of the spread of the sars outbreak in early , there was substantial international collaboration first in the isolation and identification of the cov, sharing of sequence data, and eventually the sharing of seed virus that would be suitable for vaccine development. in the case of the sanofi pasteur vaccine, the prototype virus was supplied by the centers for disease control and prevention (cdc) (utah virus p /vero cell p ). furthermore, in this spirit of collaboration, sanofi pasteur provided stocks of the vero cells for the virus isolation to better ensure that any vaccine made from these cells would be acceptable from a regulatory perspective. these were the same vero cells as are routinely used in the commercial production of inactivated polio vaccine. in addition, there was rapid sharing of immunological reagents to ensure that the plaque-purified prototype seed virus accurately reflected the circulating epidemic sars-cov. one particular difficulty with rapidly emerging infections such as sars, of course, is that they may occur anytime of the year and are unlikely to be aligned with the annual process for assigning financial and human resources and project prioritization. in order to circumvent these problems, once it became clear that the sars-cov could grow in vero cells, the national institute for allergy and infectious diseases (niaid) in the united states focused their request on several companies that had experience in large-scale vero cell culture and had the experience to work with viruses at biosafety level (bsl ) containment. our first contact with niaid on the possibility of developing an inactivated vaccine occurred in the first quarter of . sanofi pasteur was one of only two companies that satisfied the niaid's eligibility criteria. the solicitation and funding mechanism that niaid employed was a directed request for proposals (rfp). this is sometimes called a letter contract by virtue of the fact that niaid initiated contact with the companies first by letter outlining the project objectives, infrastructure requirements, and deliverables. the advantage of this mechanism, for diseases such as sars, is that in late , several hundred cases of a severe atypical pneumonia were reported in the guangdong province of the people's republic of china. by the first quarter of , similar cases were reported in hong kong and sporadically throughout south-east asia and canada. this severe disease, with a mortality of - %, spread rapidly around the world and in april , countries on different continents had reported cases [world health organization (who), accumulative sars cases]. as a result, the who issued on march a global alert for the illness that would be known as " severe acute respiratory syndrome " (sars) (who sars alert). within the same time frame, secondary cases of sars were being identified in health care workers and family members who had close contact with patients suffering from this severe respiratory illness. by the second week of june, using the who case definition (who case description), approximately sars cases and sars-related deaths had been reported to the who. while the first wave of the sars epidemic seemed to have reached its conclusion, it was completely unclear how the spread of the virus would evolve. there was no clear understanding of the animal reservoir and the impact of virus mutation and unapparent infections. different situations could be envisaged; one scenario was that virulent sarscoronavirus (sars-cov) would persist and become endemic. another possibility was that other epidemic waves would occur or, finally, that the virus would disappear. taking into account all the uncertainties and anticipating the worst-case scenario, many laboratories and vaccine manufacturers started working on a vaccine approach against sars infection, largely based on what was known from animal covs. in this chapter, we will discuss the necessity for international cooperation and the importance of discretionary funding for rapidly developing a prototype vaccine candidate. we will review the decision-making process, the strategic choices made in terms of vaccine candidate, adjuvants, working conditions, and the safety precautions implemented at the beginning and throughout the entire production process of the sars vaccine. in addition, we will discuss the unique challenges associated with moving a vaccine such as sars through the regulatory process. with such a highly pathogenic, unknown virus. we will emphasize the industrial perspectives on inactivation and decontamination experiments, the selection of the most promising vaccine candidate, the production process and the choice and use of animal models in such a pressing and difficult situation. finally, we will briefly review the unique regulatory environment created during this period for the development of a sars vaccine. the absolute eligibility criteria, vaccine characteristics, and project objectives are clearly defined; the proposal review cycle is very short; and the necessary financial resources are immediately available. in this particular case, the rfp was received by us on may , and we submitted our project plan and budget by june . preliminary work began in july and august, and by the third week of september , we had a fully executed contract. by october , our inactivated prototype sars vaccine was filled and available for clinical assessment. another positive aspect of this mechanism is that involvement of such a large research organization provides the potential to access the expertise of many different investigators, as technical problems may arise. unlike traditional research grants, these types of contracts have very short duration and, consistent with the sense of urgency, progress against objectives is monitored on a weekly basis, perhaps, contributing to the fact that our project finished almost a month ahead of schedule. importantly, this type of mechanism does not compete with academic researchers for funding but allows these researchers to develop more basic science proposals of longer duration that can be reviewed and funded by other established mechanisms. perhaps, among others, there are three important lessons about responding to emerging threats that can be learned from the sars experience. first, information must be shared quickly and transparently. the somewhat surprising observation that the sars-cov grew well in vero cells was important in prioritizing the development of an inactivated, vero cell -derived virus. second, there should be an ongoing dialogue between the national research organizations and their potential industrial partners to understand what capacity and experience could be urgently brought to bear in a crisis situation. although there are potential issues with competitive intelligence, it remains important that information about industrial capability should be shared. third, funding organizations should have in place a mechanism for rapid proposal solicitation, review and monitoring, and also adequate discretionary funding that could support new vaccine projects in an urgent manner. as early as april , , the who and the cdc in the united states (who guidelines, cdc) recommended laboratories and vaccine manufacturers to handle sars-cov specimens using standard bsl work practices. it is not exceptional for vaccine manufacturers to work in bsl facilities for the production of certain vaccines, e.g., rabies, japanese encephalitits, or polio viruses. some of these commercial vaccines have been made for decades. for these vaccines, robust production processes and standard operating procedures have been put in place. the experience gained on the decontamination and inactivation of viral vaccines during the production process and the quality control (qc) are all important. moreover, the personnel working at the different stages of such a vaccine production are typically vaccinated, and revaccination procedures are in place in case of major accidents (i.e., boost after potential rabies contamination). in case of an emerging virus such as sars, the situation is completely different. although the bsl experience will be fully exploited, all processes and procedures need to be discussed, evaluated, validated, and implemented. for each emerging virus, this exercise needs to be repeated and specific conditions must to be adopted according to the unique characteristics of the new virus. consistent with the who and cdc recommendations, at sanofi pasteur live sars-cov was handled in a bsl facility. since no treatment or vaccine was available against sars, we decided to work in " bsl plus " laboratory conditions. essentially our basic level of containment was bsl incorporating several bsl working practices: clothing change before entering the facilities, shower on exit, and all material decontaminated before exit from the facilities. also, all steps, where the product was handled in open phase, were performed within class iii biological safety cabinets (csb). to prevent accidental contamination, the laboratory workers wore a positive pressure personal mask. these precautions were considered necessary taking into consideration the large amount of live virus handled ( - l) and finally turned out to be valid as the risk for contamination existed as was shown in the laboratory accidents in singapore, taiwan, and beijing in china ( senior, ; normile, ; orellana, ) . furthermore, as described below, the decontamination experiments demonstrated that the sars-cov is an extremely resistant virus. for the medical follow-up of the bsl -trained personal involved in the sars vaccine development, it was of critical importance to be able to distinguish the symptoms of respiratory distress caused by sars or other respiratory agents. it was therefore decided that the bsl laboratory workers should be selected in accordance with their immune status and would be immunized with streptococcus pneumoniae and influenza vaccines, if appropriate. in case a worker would present symptoms, a procedure had been put in place to isolate the worker using a high-efficiency particulate air (hepa) filter mask before being transported to prior to the start of the laboratory work since it is well known, used in many vaccines, and well accepted by regulatory authorities. when the laboratory work on the sars-cov vaccine development started, no data were available on the inactivation characteristics of the virus. one of the priorities was to identify the conditions to fully inactivate the virus for vaccine development, but also for decontamination of equipment, facilities, and waste decontamination. the results, as described in more detail below, were unexpected. the sars-cov is an extremely resistant virus and several of the routine decontamination working practices cannot be applied for this virus. these results demonstrate how important it is to immediately perform decontamination testing and adapt decontamination practices and strategies for each specific (emerging) virus. at the time the development of a vaccine against an emerging virus is initiated, it is very unlikely that routine tests and reagents are available. this was indeed the case for sars. therefore, our first experiments were dedicated to develop routine tests that are required for vaccine development and the analysis of the host response after immunization. these include neutralization tests, enzyme-linked immunosorbent assays (elisa), polymerase chain reactions (pcrs), and others. well-validated reagents are needed as reference standards for essential laboratory tests as polyclonal and monoclonal antibodies, recombinant proteins, and pcr primer pairs. one of the most sensitive issues was how to select an appropriate animal model to evaluate the candidate vaccines. for example, we observed that nmri mice gave a very heterogeneous response whereas balb-c or c bl/ j mice responded uniformly to immunization with the vaccine candidate. also, guinea pigs appeared to be a good model for the evaluation of immune responses. beyond immunogenicity, it is important to work with an animal model that is appropriate for challenge studies, as an assessment of vaccine efficacy. this is especially important for emerging infections since efficacy studies in humans may not be possible. in case of the sars, the macaca fascicularis was identified very early on ( fouchier et al., ) as a likely predictive, challenge model. all of the work was done in constant communication with the regulatory authorities. our experience with sars reinforces the idea that manufacturers should be encouraged to open and maintain an active dialogue with regulatory officials, very early in the development process. a nearby hospital where a special, dedicated negative pressure hospital room had been prepared. the very first decision concerned the choice of vaccine immunization strategy. in the intense early days of the epidemic a variety of approaches were considered. these included inactivated vaccines, subunit products, dna (either alone or in combination as part of a prime-boost strategy), vectored vaccines, and live attenuated candidates. similarly, alternative routes of vaccine administration (inoculation, aerosol, etc.) and formulation (adjuvants, etc.) were considered. in fact, several live attenuated and killed vaccines for veterinary covs are already marketed: e.g., a live attenuated and killed vaccine against the chicken infectious bronchitis virus ( ladman et al., ) , a live modified virus against canine cov ( pratelli et al., ) . ultimately, however, the crucial factor in the decision-making process was likely development timelines. at the time of this decision, the epidemic was prevalent and there was a sense of urgency that a prototype vaccine had to be developed as soon as possible . embedded in this decision-making process was the realization that we were seeking a vaccine candidate that largely used existing, conventional technology and that could be made in significant quantities, if necessary. in the context of the likelihood for rapid development and the possibility for large-scale production, the decision was taken to produce a monovalent, whole, inactivated, aluminum-adjuvanted sars vaccine for intramuscular injection. from our perspective, a whole, inactivated vaccine was a logical first choice taking into consideration our extensive experience with inactivated vaccines such as inactivated poliovirus, rabies, and hepatitis a vaccines. we were also encouraged by the fact that sars-cov grew very well in vero cells. sanofi pasteur has a long industrial experience with these particular cells and developed cell banks that are validated and registered around the world. even for such a direct experience-based approach, we realized that it would take months to make a good manufacturing practice (gmp) clinical lot for phase and clinical studies. killed vaccines need, in many cases, adjuvantation. in our case, the adjuvant of choice was aluminum hydroxide. again, in the situation where a new virus is emerging, there is no time to evaluate new adjuvants. our preference was to use aluminum hydroxide viruses can be inactivated by several methods, based on either physical or chemical mechanisms. we investigated five decontamination methods that are currently used for equipment, facilities, and waste decontamination: heat, alkaline treatment, sodium hypochlorite treatment, gaseous formol fumigation, and drying. it should be stressed that the data presented here have been obtained under our specific experimental conditions. indeed, the virus sensitivity to inactivation depends on the virus environment and concentration. thus, the methods presented here were validated with our specific suspensions and experimental conditions, and appropriate precautions should be taken when manipulating sars-cov under other laboratory conditions. inactivation experiments using different ph values gave rise to unexpected results. when the ph was adjusted to ph or ph . , a strong decrease in the viral infectivity titer can be observed. however, the virus is not totally inactivated and even after h of alkaline treatment, infectious particles can still be observed. total inactivation is observed only after h of treatment. these data are surprising as enveloped viruses are usually sensitive to such drastic alkaline conditions. the results from the experiments performed to evaluate the viral loss of the sars-cov due to drying on glass surface were also surprising: - days were necessary to inactivate the virus to the detection limit of the technique. other viruses, such as polio or rabies, can be inactivated by drying durations of approximately and h, respectively. in our experience, the sars-cov is the most resistant virus ever described in an industrial setting. the gaseous formaldehyde fumigation is a viral decontamination technique widely used throughout the world. we found that formol fumigation is totally inefficient on dried virus. however, virus in solution is efficiently inactivated. the two decontamination techniques tested here, drying and formaldehyde fumigation, reinforce the necessity to decontaminate the working areas in the laboratory, as well as the equipment, very frequently in order to avoid any drying of viral suspension onto glass or any other surface. finally, sodium hypochlorite ( ° cl) and heat treatment were evaluated. the effect of sodium hypochlorite on dried virus is very rapid and efficient, as no infectious viral particles were recovered after washing the surface with sodium hypochlorite. thermal decontamination was shown to be efficient at both and ° c. to achieve total inactivation of the sars-cov, h heating at ° c and h heating at ° c are necessary. considering the decontamination data, the following strategy was put in place. all solid waste, as well as the equipment that could resist such a drastic treatment, was autoclaved. for liquid waste, the solutions were subjected to alkaline ph treatment for at least h and then transferred into a tank for thermal decontamination at ° c for min. the laboratory facilities, and the equipment that could not be autoclaved, were decontaminated with ° cl sodium hypochlorite solutions, before being fumigated with gaseous formol. the data on decontamination of the sars-cov presented here show that existing decontamination strategies cannot be directly extrapolated to emerging viruses and that these inactivation conditions should be determined empirically for each virus. the sars-cov seed virus isolate was provided on august , , by the cdc. this isolate (the so-called utah strain) was made from the sputum from an acutely ill u.s. traveler who had apparently been exposed in hong kong. this isolate was fully sequenced by the cdc and shown to be virtually identical to the urbani strain of sars-cov. to obtain an original seed virus, in full accordance with food and drug administration (fda) requirements, sanofi pasteur provided certified vero cells to the cdc, who performed the isolation of the virus and made two passages before sending the virus to sanofi pasteur. upon receipt, the virus underwent two additional passages, and was plaque purified. this plaque purification step is of importance to limit the risk of adventitious agents during the subsequent expansion of the virus. in contrast, there is also a risk that in selecting a plaque, it may differ significantly from the uncloned vaccine. to evaluate the latter, it was decided to compare cloned vs. uncloned virus in terms of virus sequence and immunogenicity in guinea pigs. it was demonstrated that the two candidate vaccines were totally similar. following this verification, the selected clone (vvnfl ) was passaged eight additional times for adaptation. from our experience with viruses to be used to prepare an inactivated viral vaccine at industrial scale, there is a need to reach a titer of virus Ͼ . log/ml. indeed, with the sars-cov, we obtained a consistent titer of around . log tcid /ml. in vero cells, distinct cytopathic effect (cpe) is always observed at day or post-infection. these first characterization was performed using pcr testing. the pcr tests are listed in table . . all tests were performed in accordance with international requirements, showing that no adventitious agents were detected. the difficulty with inactivating viruses remains the balance between fully validated inactivation and preservation of immunogenicity or epitopes associated with protection. it is well known that reagents used to inactivate viruses [betapropiolactone (bpl), formaldehyde] can change the outer membrane antigens with the risk of a reduced immunogenicity of the vaccine. for the inactivation of sars-cov, we chose to test bpl. assays were performed to determine the best bpl concentration for inactivation while maintaining a good immune response in mice. virus experiments encouraged us that the prototype vaccine could be produced in vero cells using a single harvest totally compatible with our experience with inactivated poliovirus. raw materials used to develop the candidate vaccine (serum and trypsin) were selected in accordance with current regulation. calf serum was imported from australia, and gamma irradiated prior to use. the trypsin was from porcine origin and also gamma irradiated. extensive evaluation for adventitious agents was performed on the raw material, which included the search for cytopathogenic agents, hemadsorbing agents, and specific viral contaminants as bluetongue, reovirus, rabies, parainfluenza type , specific bovine viruses [adenovirus, parvovirus, respiratory syncytial virus (rsv)], bovine viral diarrhea, rhinotracheitis virus), and porcine viruses (parvovirus, adenovirus; transmissible gastroenteritis virus; hepatitis e virus, rabies virus, and porcine pestis virus). the viral seed lots were produced in vero cells. qc testing is a major step in the qualification of such a seed. of all the different tests performed (identification of the vero cells, sterility, mycoplasma, titer, and contaminating viruses), the research of contaminating virus(es), also called adventitious agents, represents the most crucial step. to detect adventitious agents, sensitive cell culture monolayers of cv- cells, human diploid mrc- cells, and chick embryo fibroblasts (cefs) were inoculated with the crude viral suspension and are observed for induced cpe and/or hemadsorption. cv- cells are used in these tests, as they are of the same species and the same origin as the cells used for vaccine manufacturing. mrc- cells are human diploid cells and can potentially reveal other viruses able to infect human cells. and finally, taking into consideration the origin of the specimen (pulmonary syndrome) we added cef, which are known to be sensitive to the infection of several respiratory viruses such as influenza virus and rsv. at the same time, adventitious agent testing was performed on control cells (search for hemagglutining or hemadsorbant viruses) and on the supernatant of the control cells (search for adventitious agents) by inoculation of the three cell lines: vero, mrc- , and cef. adventitious agent testing was also done in vivo by inoculation in suckling mice, mice, and guinea pigs, as well as the allanto ï c cavity and yolk sac of embryonated chicken eggs. complementary to the conventional methods to qualify viral seed, as described above, extensive inactivation was performed using three different bpl concentrations: / , / , and / (v/v). in our experience, a / dilution of bpl was the optimal concentration for the inactivation of sars-cov based on a balance between total inactivation and maintenance of antigenic properties. the / bpl dilution is similar to what is used for rabies vaccine inactivation. the usual way to measure viral inactivation is by kinetic studies, i.e., reduction in virus infectivity. this technique has a detection limit of . log tcid / ml. the kinetics of inactivation was performed at , , min, and , , , , , , and h, and it was shown that inactivation below the limit of detection was obtained after h. in order to increase the detection sensitivity, an amplification test was also performed. for this amplification, vero cells are incubated with a portion of the viral solution following inactivation for different periods of time. after days of incubation, the cells are trypsinized and cultivated for additional days. at the different stages of amplification, the cells were microscopically observed for cpe and at the end of the incubation (day ) an immunocolorimetric assay test was performed. our data demonstrated that the virus is fully inactivated by h, not h of bpl treatment, demonstrating that the amplification test is much more sensitive. to complete the validation of the amplification test, the minimum limit of detectable infectious viral particles was determined. this was done by spiking the inactivated vaccine with different concentrations of live virus and incubating with vero cells. using this approach, it was possible to establish the minimum virus detection as pfu. based on these data, it was concluded that inactivation with / bpl dilution for h fully inactivates the sars-cov batches. to ensure a very large safety margin, we adopted an inactivation period of h for the sars-cov vaccine production process. since a direct efficacy trial in humans will be impossible, because of a lack of naturally circulating sars-cov, the licensure of a sars-cov vaccine will depend on surrogate markers. recently (as described below) the fda adopted the animal efficacy rule that envisions that under such circumstances, demonstration of efficacy can be performed in two animal models. for sars-cov vaccine development, monkeys and ferrets can be used to evaluate candidate vaccine. both animal models show pathology in the lungs upon autopsy. the immunogenicity of the sars vaccine was evaluated in nonhuman primates, m. fascicularis, and ferrets. both animal models are susceptible to infection, do show some signs of disease (lethargy), and show signs of pulmonary lesions upon histological examination ( fouchier et al., ; martina et al., ; ter meulen et al., ; rowe et al., ; mcauliffe et al., ) . different doses of the sars vaccine ( or log tcid /ml) were injected in the presence or absence of aluminum hydroxide. two intramuscular injections were performed at a one month interval. regarding the humoral response, sustained levels of elisa and serum-neutralizing virus-specific antibodies were elicited in vaccinated monkeys and ferrets. a significant dose -effect relationship could be demonstrated. moreover, a strong adjuvant effect of aluminum hydroxide was evidenced for each vaccine dose and proved in most cases to be highly significant. in order to evaluate the efficacy of the sars vaccine, immunized monkeys and ferrets were challenged intratracheally with a heterologous hong kong sars-cov strain (coronovative, rotterdam, the netherlands). monkeys immunized with or log of inactivated virus were protected as measured by rt-pcr and viral titration on lung samples five days post-challenge. the ferrets were protected at the lower immunization dose of or log . based on our experience to date, the inactivated, adjuvanted sars-cov prototype vaccine seems to be a good candidate for further evaluation in phase studies. as with other vaccines, vaccines for sars and other emerging threats need to follow a structured pattern of regulatory development. the initial stages would be very similar to those followed for vaccines under development for conventional infectious diseases. in the united states, the earliest stages would include the development of sufficient preclinical information about the vaccine to allow the preparation of an investigational new drug (ind) application for submission to the fda (see chapter ). the ind may have information unique to the vaccine candidate but should include information about the rationale for the vaccine design, the source of the virus and other components, the manufacture of the active vaccine component, formulation, preliminary characterization of the vaccine to determine the effectiveness of the vaccine. for these agents, it would be too dangerous to conduct challenge studies in humans and the prevalence of the disease is either nonexistent, sporadic, or too small to allow the development of a reasonable clinical protocol. in anticipation of this problem, largely in the face of potential bioterrorist agents, in , the fda adapted the so-called animal rule [see federal register, may , (volume , number ) ]. under this guidance, new drugs or biological products that are intended to prevent serious or life-threatening conditions may be approved on evidence of effectiveness derived from appropriate studies in animals and any additional supporting data, if controlled clinical studies cannot be conducted in human volunteers and field trials are not possible. in order to satisfy this alternative mechanism, however, several criteria must be met. first, there is a reasonably well-understood pathophysiological mechanism that can be ameliorated or prevented by the product. second, the effect is demonstrated in more than one animal species, unless it is demonstrated in a single species that represents a sufficiently well-characterized animal model. third, the animal study endpoint is clearly related to the desired benefit in humans. and finally, the data are sufficiently well understood to allow selection of an effective dose in humans. it is therefore reasonable to expect that the effectiveness of the product in animal model(s) is a reliable indicator of its effectiveness in humans. obviously, it is too early to know whether the sars vaccine candidate as described in this chapter will move forward and be able to meet all of the criteria of the animal rule. in particular, sars vaccine development is hindered by relatively little information about human covs in general. until the rapid emergence of sars, most of the basic research was focused on animal covs and our inactivated sars vaccine candidate described in this chapter is exclusively based on experience with vaccines to animal covs. certainly, it is too early to conclude whether the ferret and/or m. fascicularis is/are the most appropriate model(s) for human sars infections. as a result, except for clinical cases documented during the outbreak, there is relatively little information about sars pathogenesis and correlates of immunity. another difficult aspect is that a feline infectious peritonitis (fip) vaccine was actually harmful to the health of the immunized cats upon challenge with wild-type fip virus ( weiss and scott, ) . before moving forward with approval, therefore, it will be very important to determine whether these adverse outcomes can be prompted or mimicked by any of the sars vaccine candidates. including purity and potential contaminants, immunological testing, and animal testing, including toxicology. even at this early stage, the vaccine should be made under gmp conditions and other laboratory work conducted under good laboratory practice (glp) conditions, as appropriate. the ind application should also include important information about the phase clinical design, focusing on how the safety will be monitored and a discussion of any potential adverse reactions based on the experience with vaccines that have similar components or methods of preparation. there should be an opportunity to outline the vaccine concept and phase clinical study at a pre-ind meeting that often provides the opportunity to receive the input and concerns of the regulatory agency. following approval of the ind, vaccines such as sars can progress to a conventionally designed phase study. typically, this phase clinical study is descriptive and would include a small number of healthy young adults with the emphasis on monitoring the safety of the vaccine (local and systemic reactions). often, the first immunologic assessment is part of this study. following successful completion of the phase , as with other vaccines, a sars vaccine candidate could move forward to phase . during this phase, in addition to safety monitoring, dose-ranging studies are conducted in much larger groups of individuals and the vaccine should meet predefined primary and secondary endpoints. if the phase is successful, following a pre-phase meeting, clinical studies are conducted in a greater number of subjects during which less frequent reactions can be detected and the efficacy or effectiveness of the vaccine determined. as part of this phase evaluation, the consistency of sequential lots of the vaccine are typically compared in order to ensure that the vaccine can be reproducibly manufactured. obviously, as the vaccine progresses clinically from phase to phase , the size of the lots of vaccine often increases and the manufacturing and in-process and release testing specifications become increasingly well defined, so as to guarantee that the vaccine can consistently be made at a commercially useful scale. if all three phases of clinical development are successful, the manufacturer may then submit a biologics license application (bla), which is a very extensive compilation of all of the information relating to the development and manufacture of the vaccine. as suggested in the animal models section above, the unique challenge for sars and other emerging threats, whether anthrax or ebola viruses, is that it may not be possible to conduct phase clinical studies the production of a gmp clinical lot of a monovalent, whole, inactivated, aluminum hydroxideadjuvanted sars-cov vaccine took months. in terms of vaccine development, this is extremely rapid. several factors contributed to these short timelines. the grants made available by the niaid for the development of a sars vaccine completely changed the classical environment, allowing vaccine industries to start almost immediately the development of a new vaccine. indeed, the development of a new vaccine can only be done to the detriment of other vaccine developments, mobilizing teams and facilities. from a technical point of view, the choice of a classical vaccine development strategy using conventional procedures, such as vero cell culture for viral propagation and bpl inactivation, was a decisive factor to success. importantly, we were able to quickly recruit a volunteer workforce that was both familiar with the technology and trained to work in a bsl -plus environment. a close collaboration with the reference laboratory, the cdc's influenza branch, where the sars-cov was isolated, was essential. we provided certified vero cells to the cdc, which allowed us, upon receipt of the purified strain from the cdc, to re-isolate the sars-cov under conditions making the prompt start of a vaccine development possible . when initiating vaccine development against a new emerging infectious agent, the problem of availability of reagents and routine tests to perform biological and molecular studies must be addressed. it is obvious, that at the beginning of such development, there are no such reagents or commercial kits available. as a consequence, the first step in the sars-cov project was to prepare the different reagents (antisera and monoclonal antibodies) and the appropriate tests (viral titration, pcr, elisa, immunofluorescence assay, etc.). finally, a series of preliminary experiments on monkeys (three months after the start of the project) had given guidance whether it was appropriate to use an inactivated vaccine, as well as to the choice of the adjuvant. constant communication with regulatory authorities has allowed the validation of this strategy from the beginning of the project. this communication was also very important for the qualification of the viral seed lots. the qualification of the vero cells was not an issue as several vaccines are already produced in vero cells, but this was obviously not the case for the viral seeds. two major obstacles had to be overcome: ( ) the realization that the animal testing had to be done in bsl facilities by bsl -trained personnel, and ( ) the search for adventitious agents using general classical tests (search for adventitious viruses on cells and in animals) and specific tests (pcr). for the latter, there was no list available and the final testing to be performed was under the responsibility of health authorities. this resulted in a rather exhaustive list of pcr testing. it is likely that epidemics will emerge in the future from unrecognized sources and some of these will be highly pathogenic for humans. these pathogens will be categorized as bsl or bsl pathogens needing high security level laboratories as well as specialized personnel. how to manipulate these pathogens that are highly pathogenic, in large quantities? to face the emergence of new pathogens, dedicated structures are needed with the right equipment and trained personnel. from an industrial perspective, this seems not compatible with the need and use of trained personnel and facilities that do have a constant activity to assure the production of existing vaccines and the development of new vaccines. such emergency structures could be set up and maintained by national reference centers respecting the bsl requirements as well as the gmp conditions. it would be very beneficial for industries to collaborate with such reference centers that provide purified pathogens and reagents allowing a prompt start of a vaccine development . aetiology: koch's postulates fulfilled for sars virus protection of chickens after live and inactivated virus vaccination against challenge with nephropathogenic infectious bronchitis virus pa/wolgemuth/ virology: sars virus infection of cats and ferrets replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys infectious diseases. second lab accident fuels fears about sars laboratory-acquired sars raises worries on biosafety safety and efficacy of a modified-live canine coronavirus vaccine in dogs macaque model for severe acute respiratory syndrome recent singapore sars case a laboratory accident human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever key: cord- -gkm i s authors: yamada, yoshiyuki; liu, xiao bo; fang, shou guo; tay, felicia p. l.; liu, ding xiang title: acquisition of cell–cell fusion activity by amino acid substitutions in spike protein determines the infectivity of a coronavirus in cultured cells date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: gkm i s coronavirus host and cell specificities are determined by specific interactions between the viral spike (s) protein and host cell receptor(s). avian coronavirus infectious bronchitis (ibv) has been adapted to embryonated chicken eggs, primary chicken kidney (ck) cells, monkey kidney cell line vero, and other human and animal cells. here we report that acquisition of the cell–cell fusion activity by amino acid mutations in the s protein determines the infectivity of ibv in cultured cells. expression of s protein derived from vero- and ck-adapted strains showed efficient induction of membrane fusion. however, expression of s protein cloned from the third passage of ibv in chicken embryo (ep ) did not show apparent syncytia formation. by construction of chimeric s constructs and site-directed mutagenesis, a point mutation (l -f) at amino acid position in the heptad repeat region of s protein was shown to be responsible for its acquisition of the cell–cell fusion activity. furthermore, a g -d point mutation in the s domain, which was acquired during further propagation of vero-adapted ibv in vero cells, could enhance the cell–cell fusion activity of the protein. re-introduction of l back to the s gene of vero-adapted ibv allowed recovery of variants that contain the introduced l . however, compensatory mutations in s and some distant regions of s were required for restoration of the cell–cell fusion activity of s protein carrying l and for the infectivity of the recovered variants in cultured cells. this study demonstrates that acquisition of the cell–cell fusion activity in s protein determines the selection and/or adaptation of a coronavirus from chicken embryo to cultured cells of human and animal origins. interspecies adaptation, replication and transmission in cells are essential steps for an animal virus to emerge successfully in a human population. virus-cell and cell-cell membrane fusion, mediated by fusion proteins associated with viral envelope, is crucial for the entry of enveloped viruses into cells and for rapid spread of infection to the neighboring cells. this membrane fusion process may, therefore, be a limiting point for efficient adaptation and infection of an animal virus in cells from a different host species. in this study, we report that acquisition of the cell-cell fusion activity by point mutations in the spike (s) protein of avian coronavirus infectious bronchitis virus (ibv) plays a critical role in adaptation and/or selection of a variant that infects cultured cells. coronavirus is a large family of enveloped, positive-stranded rna viruses that cause respiratory and intestinal infections in avian and mammalian species [ ] . ibv, the prototype member of coronavirus, causes highly contagious diseases in chicken and is a constant threat to the poultry industry. coronavirus was traditionally considered to have narrow host specificities [ ] . however, the outbreaks of severe acute respiratory syndrome (sars), a serious zoonotic transmission event caused by a novel coronavirus, demonstrate that a certain coronvirus species may exhibit wider host specificities and suggests the possibility of crossspecies transmission of animal coronaviruses to human [ , ] . cross-species transmission was also observed in coronavirus transmissible gastroenteritis virus (tgev) and human coronavirus oc [ ] [ ] [ ] . these events highlight the importance of understanding the mechanisms of interspecies adaptation and transmission of coronavirus. the beaudette strain of ibv was previously adapted to embryonated chicken eggs. this embryo-adapted ibv strain was subsequently adapted to cultured cells originated from chicken and monkey. for example, the virus was adapted by serial passages to primary chicken kidney (ck) cells [ , ] and the african green monkey kidney cell line vero cell [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, the veroadapted ibv is able to infect cultured human and animal cell lines [ , ] . in a previous report, a total of amino acid substitutions was found during adaptation of ibv from chicken embryo (ep ) to vero cells (p ) [ ] [ ] [ ] . among them, amino acid substitutions are in the s protein [ ] . in this study, expression of s protein cloned from ibv strains ep , ck, passage (p ) and p of vero-adapted ibv showed induction of cell-cell fusion by s(ck), s(p ) and s(p ) constructs. however, no formation of syncytial cells was observed in cells expressing s(ep ). construction of chimeric s constructs and site-directed mutagenesis studies identified a leucine to phenylalanine substitution at the amino acid position in the heptad repeat region (l -f) that confers the non-fusogenic s protein to fusogenic. re-introduction of the f -l mutation back to the genome of vero-adapted ibv (p ) showed rescue of virus containing the f -l mutation. however, compensatory mutations occurred in the s region that could rescue the cell-cell fusion activity of s constructs carrying the f -l mutation. cells were maintained in dmem supplemented with % newborn calf serum. the vero-adapted ibv and recombinant vaccinia/t virus was propagated and titrated on vero cells. virus stocks were kept at uc until use. cell monolayers grown on well slide chambers were infected with vaccinia/t virus for hour followed by transfection of indicated plasmids using the effectene transfection kit (qiagen). at hours post-transfection, cells were washed with phosphate buffered saline (pbs) supplemented with % normal goat serum, fixed with % paraformaldehyde in pbs for minutes, and permeabilized with . % triton x- . immunofluorescent staining was performed by incubation of cells with rabbit anti-ibv s polyclonal antibodies and subsequently with fitcconjugated anti-rabbit igg. cells were examined by fluorescent microscopy and digital images were collected. protein samples were prepared from cells harvested at hours post-transfection, separated by sds-page and transferred to pvdf membranes. the membranes were incubated with rabbit anti-ibv s polyclonal antibodies or mouse anti-b-tubulin monoclonal antibody (sigma aldrich), and subsequently with hrp-conjugated anti-rabbit or -mouse igg (dako). polypeptides were detected using the enhanced chemiluminescence (ecl) detection reagents (amersham). vero cells were transfected as described above, and harvested at hours post transfection. cells were washed once with pbs, resuspended in blocking buffer containing % fbs and % bsa in pbs, and incubated on ice for minutes. subsequently, cells were incubated with . % saponin in facs washing buffer containing . % fbs and . % sodium azide in pbs for minutes at room temperature when required. immunofluorescent staining was carried out with : diluted rabbit anti-ibv s polyclonal antibodies, and : diluted fitc-conjugated swine anti-rabbit antibody (dako). after washing two times with the facs washing buffer, cells were fixed with % ice cold paraformaldehyde and analyzed by flow cytometry. viral rna was extracted from the culture supernatants or infected cells using the rneasy mini kit (qiagen) according to the manufacturer's instructions. rt-pcr was performed using the expand reverse transcription and high fidelity pcr kits (roche). the pcr products were cloned into pcrh-xl-topoh vector (invitrogen) and sequenced by automated sequencing. construction of the full-length ibv cdna clones from p of vero-adapted ibv was previously reported [ , ] . the f -l point mutation was introduced into the corresponding fragment using quickchange site-directed mutagenesis kit (stratagene), and subsequently ligated into the full-length cdna clone. the full-length transcripts were generated in vitro using the mmessage mmachine t kit (ambion) according to the manufacturer's instructions with certain modifications. briefly, ml of transcription reaction with a : ratio of gtp to cap analog were sequentially incubated at . uc for minutes, . uc for minutes; . uc for minutes, and . uc for minutes. the transcripts were extracted with phenol/chloroform. vero cells were grown to % confluence, trypsinized, washed twice with cold pbs, and resuspended in pbs. rna transcripts were added to ml of vero cell suspension in an electroporation cuvette, and one electrical pulse at v, mf was given using a bio-rad gene pulser ii electroporator. the transfected vero cells were cultured overnight in % fbs-containing mem in a mm dish or a six-well plate and further cultured in mem without fbs. the s genes from different passages of the vero-adapted ibv strain and ck-adapted ibv were amplified and cloned into pkt vector [ ] . chimeric s constructs were made by overlapping pcr [ ] . point mutations were introduced by site-directed mutagenesis using the quikchange tm kit (stratagene). all constructs were confirmed by automated nucleotide sequencing. cell-cell fusion activity of s proteins cloned from ep and ck-adapted beaudette strain of ibv acquisition of the cell-cell fusion activity is essential for selection and adaptation of coronavirus ibv from chicken embryo to cultured cells [ ] . sequence comparison of two s protein constructs, s(ep ) and s(ck), cloned from ep and ck-adapted ibv strains, respectively, showed amino acid substitutions at positions (fig. a) . the cell-cell fusion activity of these two s constructs was analyzed by transfection into vero cells using the vaccinia/t recombinant virus system. western blot analysis showed the presence of major forms of s protein, including the -kda glycosylated (s*) and -kda unglycosylated full-length s (s) and the cleaved s and s species (s /s ) in cells expressing the two constructs (fig. a, lanes and ) . it was noted that the expression level of s(ck) was higher than s(ep ) (fig. a) . as a negative control, cells transfected with ibv n protein were included, and the expressed n protein was detected by western blot with anti-n antibodies (fig. a, lane ) . immunofluorescent staining of vero cells expressing s(ck) clearly showed syncytia formation at hours post-transfection (fig. b , panel s(ck)). however, in vero cells expressing s(ep ), no obvious syncytia was observed (fig. b , panel s(ep )). in the negative control cells, no fusion of the transfected cells was detected (fig. b, panel n) . to investigate the possibility that intrinsic differences in cell surface translocation of the two s constructs may affect their cellcell fusion activity, cell surface expression of the two proteins was analysed by flow cytometry after immunofluorescent staining with anti-s antiserum. as shown in fig. c acquisition of the cell-cell fusion activity by mutation of a conserved leucine residue to phenylalanine (l -f) in the heptad repeat region of s protein to map the amino acid mutation(s) responsible for acquisition of the cell-cell fusion activity of s(ck), three chimeric constructs were first made. construct ep -ck( ) was made by replacing the c-terminal amino acid region of s(ep ) with the corresponding region from s(ck), ep -ck( ) was made by replacing the cterminal amino acid region of s(ep ) with the corresponding region from s(ck), and ck-ep was made by replacing the nterminal amino acid region of s(ep ) with the corresponding region from s(ck) (fig. a) . western blot analysis of cells expressing these constructs detected the s and s species as well as the glycosylated and unglycosylated forms of the full-length s protein (fig. b , lanes [ ] [ ] [ ] . immunofluorescent staining showed cell-cell fusion and syncytia formation in cells expressing both ep -ck( ) and ck-ep (fig. c , panels ep -ck( ) and ck-ep ), but not ep -ck( ) (fig. c , panel ep -ck ( )). the relative cell-cell fusion activities of these s constructs were semiquantitatively defined by comparing the average size of syncytia induced by different s constructs with the average size (considered as ) of cells expressing s(ep ), and are listed in the order from high to low as follows: ck-ep .ck = ep -ck( )&ep = ep -ck( ) (.indicates the relative activity is within fold higher, and &indicates more than fold higher). these results demonstrate that the region between amino acids and may determine the fusogenic difference between s(ep ) and s(ck). examination of this region showed two amino acid substitutions from s(ep ) to s(ck), i.e. n to s and l to f (fig. b) . to determine which amino acid substitution dictates the fusogenic change, three mutant constructs were made. constructs ck (s -n) and ck(f -l) were made by mutation of the s and f residues in s(ck) to n and l, respectively (fig. a) . construct ep (l -f) was made by mutation of the l residue in s(ep ) to f (fig. a) . western blot analysis of cells expressing these constructs detected the s and s species as well as the fulllength forms (fig. b , lanes - ). immunofluorescent staining showed formation of syncytia in cells expressing ck(s -n) (fig. c , panel ck(s -n), suggesting that mutation of s to n did not affect the cell-cell activity of s(ck). cell-cell fusion and syncytia formation were also observed in cells expressing ep (l -f) but not ck(f -l) (fig. c , panels ep (l -f) and ck(f -l)), demonstrating that the l -f mutation introduced into s(ep ) renders the protein fusogenic in cultured cells. on the other hand, mutation of the f residue to l totally abolishes the fusion activity of s(ck) (fig. c , panel ck(f -l). the relative cell-cell fusion activities of these s constructs are ck(s -n).ep (l -f)&ep = ck(f -l). these results confirm that s(ck) gains the cell-cell fusion activity by l -f mutation in the heptad repeat region of the protein. the f -l substitution was then introduced into the s constructs cloned from vero-adapted ibv (p ) and (p ), respectively, generating s(p ) and s(p ) (fig. a) . expression of these constructs showed cell-cell fusion and syncytia formation in cell expressing wild type s(p ) and s(p ), but not the mutant s proteins (fig. c , panel p , p (f -l), p and p (f -l)). the expression levels of both f -l mutants were lower than the wild type constructs, but no significant difference in the s /s cleavage was observed (fig. b, lane - ). the relative cell-cell fusion activities of these s constructs are p .p &ep = p (f -l) = p (f -l). these data indicate that s protein acquires its cell-cell fusion activity by the l -f mutation during adaptation to both ck and vero cells. further mutations of the l residue to other amino acids based on s(ep ) were made. as shown in fig. a , the l was mutated to y, s, e, i and k, respectively. expression of these mutant constructs showed that mutations of l to y and s exhibited similar effect on cell-cell fusion as the l -f mutant. cell-cell fusion and syncytia formation were observed in cells expressing these two mutants (fig. c) . however, much less cell-cell fusion and smallersized syncytia were observed in cells expressing l -e, l -k and l -i mutant constructs (fig. c) . the relative cell-cell fusion activities of these s constructs are ep (l -y).ep (l -s).ep (l -e).ep (l -i) = ep (l -k).ep . introduction of the f -l substitution back to the genome of vero-adapted ibv and analysis of its effect on viral infectivity in cultured cells the f -l mutation was then introduced back to the genome of vero-adapted ibv by using an infectious clone system based on p [ , ] to test its influence on viral recovery and infectivity. in vitro synthesized full-length transcripts derived from wild type (ribv) and mutant (fl) clones were introduced into vero cells by electroporation. at days post-electroporation, syncytia formation the recombinant wild type and mutant viruses (p ) were recovered from the culture media at and days post-electroporation, respectively, and further propagated on vero cells for passages. total rna was extracted from the culture media of cells infected with each passage of the mutant virus and rt-pcr was carried out to amplify the s gene. the rt-pcr products were cloned, bacterial clones were randomly chosen from p , and the complete nucleotide sequence of the s gene was determined to confirm if the recovered virus maintains the f -l substitution. as shown in table , l was found in all clones. however, only five clones had an identical sequence with the original mutant s gene (type fl), and additional mutations at other positions were found in the other five clones (table ) . among them, two clones contain a t -s substitution (flv ), one contains an i -v substitution (flv ), and two contain q -l and i -v substitutions (flv ) ( table ). these results demonstrate that the recovered fl mutant virus from p contains a mixed population of quasispecies. to investigate which quasispecies would become dominant in the subsequent passages, sequencing analysis of bacterial clones containing the pcr fragments from p , p and p was performed. in the four clones chosen from p , a homogenous s gene with both q -l and i -v (flv ) mutations was found (table ) . subsequent sequencing of clones derived from p and p each showed that six out of clones from p and two out of clones from p are flv (table ). the dominant clones contain an additional proline to serine substitution at amino acid position (flv ) ( table ) . the recovered viruses were then plaque-purified. compared to wild type ibv, ribv showed similar growth kinetics in vero cells (fig. b) , but formed slighlty smaller plaques (fig. a) with lower expression level of s protein (fig. c) . a total of mutant viruses was plaque-purified from passages and , and the s gene of all purified viruses was shown to share the same sequence as flv ( table ). the flv mutant virus formed similar-sized plaques as ribv (fig. a) with slightly lower expression of s protein (fig. c) . interestingly, the mutant virus produced up to -fold higher titers of virus, compared to ribv (fig. b) . restoration of the cell-cell fusion activity of s(p ) protein carrying the f -l mutation by compensatory mutations in the s region the cell-cell fusion activity of s proteins cloned from the mutant ibv construct fl and the four variants (flv , flv , flv and flv ) was analyzed by expression in vero cells. once again, expression of these constructs led to the detection of s and s species as well as the full-length forms (fig. a) . higher levels of s protein were detected in cells expressing s(flv ) and s(flv ), comparing to cells expressing the other two s constructs (fig. a) . immunofluorescent staining showed the formation of giant syncytia in cells expressing s(flv ) and s(flv ) (fig. b , panels s(flv ) and s(flv )), but much smaller syncytia were observed in cells expressing s(flv ) (fig. b, panel s(flv ) ). no obvious cell-cell fusion was observed in cells expressing s(fl) and s(flv ) (fig. b , panels s(fl) and s(flv )). the relative cell-cell fusion activities of these s constructs are flv .flv .flv &ep = fl = flv . these results confirm that acquisition of the cell-cell fusion activity is an important step for adaptation of ibv in cultured cells. since amino acid difference between s(flv ) and s(flv ) was only at the th residue, the s(fl(q -l)) construct was also created and expressed. the results showed that it displayed a similar cell-cell fusion activity as flv ( = fl(i -v)) ( fig. a further enhancement of the cell-cell fusion activity of s protein and adaptation of ibv to cell culture by g -d substitution vero-adapted ibv gradually increased its infectivity in vero cells by serial passages and a significant difference between p and p was observed [ ] . comparison of amino acid sequences between s(p ) and s(p ) revealed a single mutation at the amino acid position (g -d) in s(p ) (fig. a) . to analyze the possibility that the enhanced infectivity of p virus is due to the enhanced cell-cell fusion activity of the corresponding s protein, s(p ) and s(p ) constructs were created and expressed in vero cells. efficient induction of cell-cell fusion was observed in cells transfected with both constructs (fig. a, panels s(p ) and s(p ) and b, lanes and ) . comparatively, significantly larger syncytia was observed in cells expressing s(p ) construct than in cells expressing s(p ) (fig. a) , demonstrating that the additional g -d mutation in s(p ) may enhance its cell-cell fusion activity. the g -d mutation was then introduced into s(ep ) and s(ck) and expressed (fig. a) , showing that introduction of g -d mutation into s(ck) drastically enhanced its cell-cell fusion activity (fig. b) . interestingly, introduction of the mutation into s(ep ) and expression of the construct in vero cells showed formation of small syncytial cells (fig. b) . the relative cell-cell fusion activities of these s constructs are ck(g -d).p .ck&p .ep (g -d).ep . avian coronaviruses have been isolated from chicken, turkey and pheasant and may exist in many other avian species [ ] . ibv is usually associated with respiratory disease in domestic fowl, and was believed to have a limited host range. chicken is the only natural host. similarly, coronaviruses originated from human and other animal species were considered to have narrow host specificities until the identification of sars-cov as the causative agent of sars outbreaks in . the current model of animal origin of sars-cov highlights the importance of cross-species adaptation and transmission of animal coronaviruses to human. cross-species transmission of virus infection has long been recognized as a way for the emergence of many zoonotic diseases. the molecular basis for this phenomenon would lie on the rapid adaptation of certain viruses to a changing environment through selection of minor variants from quasispecies, accumulation of mutations, recombination between minor variants, and reassortment of their genomes. a better understanding of the underlying mechanisms that control these events would be essential for providing safeguards to limit the impact of these devastating diseases. in this study, we show that acquisition and enhancement of the cell-cell fusion activity by amino acid substitutions in the s protein are critical for interspecies adaptation and infectivity of ibv to cultured cells. data present clearly show that the l -f mutation in the heptad repeat region of s proteins derived from cell-culture-adapted ibv is important for adaptation of the virus to cell culture systems, and an additional mutation in the s region (g -d) could enhance this process. as s protein carrying the l -f mutation is able to induce cell-cell fusion, but losses the activity when f was mutated back to l, it suggests that induction of cell-cell fusion is an essential step in adaptation/ selection of ibv to cultured mammalian cells. coronavirus s protein is the major determinant for viral entry and host specificity. it is a class i fusion protein and mediates viral entry by specific binding of the s domain to a host cell receptor [ ] [ ] [ ] [ ] [ ] . the cellular receptors for several coronaviruses have been identified, including members of the cacinoembryonic antigen family of cell adhesion molecules as the receptor for mhv, angiotensin converting enzyme ii for sars-cov and human coronavirus nl , and aminopeptidase n for human coronavirus e and tgev [ ] [ ] [ ] [ ] [ ] . to date, the receptor(s) for ibv has not been identified in its native or adapted host cells. it is assumed that a mammalian counterpart on vero cells could be used as a receptor for ibv at low affinity, and might have structural and functional similarities to the native receptor on chicken cells. at the initial stages of the adaptation process, a certain proportion of ep would weakly bind to this molecule and gains entry into the cells by endocytosis. in addition, binding of ibv to sialic acid was reported to be important for adaptation of the virus to human cells [ , ] . the beaudette strain of ibv was also reported to have an additional binding activity to heparin-like structures [ ] . these additional binding activities may help to initiate infection and thus allow the virus to adapt to the new host receptor by mutation. to uncoat the engulfed virion and to establish subsequent infection cycles as well as to spread infection to neighboring cells, acquisition of virus-cell/cell-cell membrane fusion and enhancement of the cell-cell fusion activity would be an essential step for successful selection/adaptation of virus to the new host cells. membrane fusion mediated by coronavirus, similar to other viruses, is a multistep process. it includes binding of the s protein to one or more receptors, conformational changes of the protein to a fusionactive form and the actual fusion process. the membrane-fusion activity of coronavirus s protein is mainly associated with domains in the s region of the protein [ ] [ ] [ ] [ ] [ ] . in this study, we demonstrate that l -f substitution in the s region of the ibv s protein confers the s protein from non-fusogenic to fusogenic. this mutation may affect one of these fusion steps and thus modify the fusion activity of s protein and syncytia formation. at the same time, the virus was successfully adapted to the cultured cells with enhanced infectivity, confirming that acquisition of membrane fusion is an important step in selection/adaptation of ibv to cell culture and may also play a crucial role in cross-species adaptation and transmission of ibv in cultured cells. further enhancement of the cell-cell fusion activity of ibv s protein was achieved by a single amino acid substitution (g -d) in p virus. this mutation, meanwhile, enhances the infectivity of the virus in cultured cells. the enhancement effect by mutations in the s region and its significance on viral infectivity was further demonstrated by cloning and expression of s gene derived from the ibv variants rescued from the full-length transcripts containing the f -l mutation. in variants flv and flv , additional amino acid substitutions (q -l and i -v) greatly enhanced the cell-cell fusion activity of the l -containig s protein and the infectivity of the recovered virus. the involvement of residues in the s region in the cell-cell fusion activity of s protein was also demonstrated in other coronaviruses [ ] . these results illustrate the complexity of the fusion process and the involvement of multiple domains in the induction of membrane fusion. it is worth mentioning that the cell-cell fusion activity of various s constructs was approximated by the degree of cell-cell fusion induced in cells overexpressing individual constructs. in a previous report, we showed nice correlation between the cell-cell fusion activity of two s constructs and their expression levels in the cells [ ] . this correlation was also observed in this study with more wild type and mutant s constructs. based on data generated from adaptation [ ] and cell-cell fusion studies presented here, a model of two-step adaptation process is proposed (fig. ) . in this model, the adaptation was divided into primary and secondary adaptation (fig. ) . early passages of vero-adapted ibv, including p , p , p and p , belong to the primarily adapted group (fig. ) . other cell cultureadapted strains, including the ck-adapted and beaudette-us strains cac and cac [ , ] , and the vero-adapted strain aav described by youn et al. [ ] , also belong to this group (fig. ) . late passages of vero-adapted ibv, including p , p and p , contain the additional g -d amino acid substitution and belong to the secondarily adapted group (fig. ) . except in the cell culture-adapted ibv strains, the l residue was found to be absolutely conserved in all coronaviruses sequenced so far. as structural information for this ibv s protein is currently lacking [ , ] , we are unclear the overall role of this residue on the formation and stability of the six-bundle structure of the protein. further structural and functional studies are required to delineate the precise roles of this mutation in the fusion process. mutation of this residue to either a ser or a tyr showed similar effect on the cell-cell fusion activity of the s protein as a phe. on the other hand, when the residue was mutated to ile, glu or lys, a much lower cell-cell fusion activity of the s protein was observed. interestingly, mutations in the s and some distant s regions could compensate the effect of f -l mutation. this may explain why s protein from several other coronaviruses, such as mhv and human coronavirus, could induce efficient virus-cell/cell-cell fusion although a conserved leu residue was found at the equivalent position [ , ] . it is worth mentioning that the cell-cell fusion activities of different s constructs were qualitatively and semi-quantitatively determined in cells overexpressing individual s constructs using the vaccinia/t expression system. attempts to obtain more rigorous quantitative data were unsuccessful. as shown in this figure . diagram showing a two-step adaptation process of chicken embryo-adapted ibv to vero cells. also shown are the numbers of amino acid changes during each adaptation process. a the accession no. for s genes from ep is dq , p is dq , p is dq . b the accession no. for this vero-adapted strain is aav . c the accession no. for these two strains are cac and cac . doi: . /journal.pone. .g study, s protein is inefficiently translocated to the cell surface, probably due to the presence of an er retention signal [ ] . since cell surface expression of s protein is a prerequisite for the induction of cell-cell fusion, disruption of the er-retention signal may facilitate cell surface expression as well as quantitative analysis of the cell-cell fusion activity of the protein. as virus-cell/cell-cell fusion is essential for efficient propagation of viral infection, attempts to interfere this process with fusion inhibitors, such as peptides or small molecules, are being made for several viruses, including hiv, sars-cov and influenza virus. the involvement of multiple domains in the induction of cell-cell fusion demonstrated here would complicate the design of such inhibitors. furthermore, mutations in regions beyond the target sequence, in the case of coronaviruses the s and some distant s regions, may lead to the emergence of drug-resistant strains. understanding of the fusion mechanisms in more detail would, therefore, help design more efficient inhibitors. conceived and designed the experiments: dxl. performed the experiments: yy xbl sf fpt. analyzed the data: yy xbl sf dxl. wrote the paper: xbl dxl. the molecular biology of coronaviruses the biology and pathogenesis of coronavirus isolation and characterization of viruses related to the sars coronavirus from animals in southern china bats are natural reservoirs of sars-like coronaviruses murine encephalitis caused by hcov-oc , a human coronavirus with broad species specificity, is partly immune-mediated genetic evolution and tropism of transmissible gastroenteritis coronaviruses circulation of genetically distinct contemporary human coronavirus oc strains replication and morphogenesis of avian coronavirus in vero cells and their inhibition by momensin coronavirus ibv: partial amino terminal sequencing of spike polypeptide s identifies the sequence arg-arg-phe-arg-arg at the cleavage site of the spike precursor propolypeptide of ibv strains beaudette and m selection of and recombination between minor variants lead to the adaptation of an avian coronavirus to primate cells emergence of an avian coronavirus infectious bronchitis virus (ibv) mutant with a truncated b gene: functional characterization of the b gene in pathogenesis and replication single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature in vitro assembled, recombinant infectious bronchitis viruses demonstrate that the a open reading frame is not essential for replication induction of p -independent cell cycle arrest at s-and g /m-phase in cells infected with the coronavirus infectious bronchitis virus promotes viral replication identification of two new polypeptides encoded by mrna of the coronavirus infectious bronchitis virus an arginine-to-proline mutation in a domain with undefined functions within the helicase protein (nsp ) is lethal to the coronavirus infectious bronchitis virus in cultured cells amino acid residues critical for rna-binding in the n-terminal domain of the nucleocapsid protein are essential determinants for the replication and infectivity of coronavirus in cultured cells further characterization of the coronavirus infectious bronchitis virus c-like proteinase and determination of a new cleavage site membrane association and dimerization of a cysteinerich, -kda polypeptide released from the c-terminal region of the coronavirus infectious bronchitis virus a polyprotein coronaviruses from pheasants (phasianus colchicus) are genetically closely related to coronaviruses of domestic fowl (infectious bronchitis virus) and turkeys the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex murine coronavirus with an extended host range uses heparan sulfate as an entry receptor cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i a -amino acid stretch in the hypervariable region of the spike protein s subunit is critical for cell fusion activity of mouse hepatitis virus aminopeptidase n is a major receptor for the enteropathogenic coronavirus tgev human coronavirus nl employs the severe acute respiratory syndrome coronavirus receptor for cellular entry angiotensinconverting enzyme is a functional receptor for the sars coronavirus receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins human aminopeptidase n is a receptor for human coronavirus e sialic acid is a receptor determinant for infection of cells by avian infectious bronchitis virus infection of hela cells by avian infectious bronchitis virus is dependent on cell status heparan sulfate is a selective attachment factor for the avian coronavirus infectious bronchitis virus beaudette receptor-induced conformational changes of murine coronavirus spike protein n-terminal domain of the murine coronavirus receptor ceacam is responsible for fusogenic activation and conformational changes of the spike protein fusogenic properties of uncleaved spike protein of murine coronavirus jhmv the n-terminal domain of the murine coronavirus spike glycoprotein determines the ceacam receptor specificity of the virus strain conformational changes in the spike glycoprotein of murine coronavirus are induced at uc either by soluble murine ceacam receptors or by ph coronavirus spike protein inhibits host cell translation by interaction with eif f structural basis for coronavirusmediated membrane fusion. crystal structure of mouse hepatitis virus spike protein fusion core crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core amino acid substitutions and an insertion in the spike glycoprotein extend the host range of the murine coronavirus mhv-a acquired fusion activity of a murine coronavirus mhv- variant with mutations in the proteolytic cleavage site and the signal sequence of the s protein infection of the tracheal epithelium by infectious bronchitis virus is sialic acid dependent key: cord- - ti mru authors: wei, xiaona; she, gaoli; wu, tingting; xue, chunyi; cao, yongchang title: pedv enters cells through clathrin-, caveolae-, and lipid raft-mediated endocytosis and traffics via the endo-/lysosome pathway date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: ti mru with the emergence of highly pathogenic variant strains, porcine epidemic diarrhea virus (pedv) has led to significant economic loss in the global swine industry. many studies have described how coronaviruses enter cells, but information on pedv invasion strategies remains insufficient. given that the differences in gene sequences and pathogenicity between classical and mutant strains of pedv may lead to diverse invasion mechanisms, this study focused on the cellular entry pathways and cellular transport of the pedv gi and gii subtype strains in vero cells and ipec-j cells. we first characterized the kinetics of pedv entry into cells and found that the highest invasion rate of pedv was approximately % in the ipec-j cells and approximately % in the vero cells. to clarify the specific endocytic pathways, systematic research methods were used and showed that pedv enters cells via the clathrin- and caveolae-mediated endocytosis pathways, in which dynamin ii, clathrin heavy chain, eps , cholesterol, and caveolin- were indispensably involved. in addition, lipid raft extraction assay showed that pedv can also enter cells through lipid raft-mediated endocytosis. to investigate the trafficking of internalized pedv, we found that pedv entry into cells relied on low ph and internalized virions reached lysosomes through the early endosome–late endosome–lysosome pathway. the results concretely revealed the entry mechanisms of pedv and provided an insightful theoretical basis for the further understanding of pedv pathogenesis and guidance for new targets of antiviral drugs. as a type of alphacoronavirus, porcine epidemic diarrhea virus (pedv) has caused enormous economic loss to the global pork industry, especially after the emergence of highly pathogenic pedv variant strains in . pedv was first reported in in the uk [ ] , and afterward was also discovered in europe and asia [ ] [ ] [ ] [ ] . although pedv has persisted in asian swine-producing countries, it does not attract enough global attention. after october , severe ped outbreaks occurred even in chinese pig farms that were already vaccinated with cv inactivated or live-attenuated vaccines [ , [ ] [ ] [ ] . in , the first pedv outbreak occurred in the us and rapidly spread across the entire country [ ] . molecular epidemiological results show the genetic differences between classical (gi subtype) and new pedv variant strains (gii subtype) [ , , , , , ] . pedv is presently recognized worldwide due to dramatic changes observed in its epidemic character, pathogenic properties, and gene drift [ ] . studies focused on its pathogenesis [ , ] , immune evasion [ ] and developing effective vaccines [ , ] are progressing. a virus is a non-cellular life form that must rely on cells to complete its life cycle. the first step in virus infection is successful entry into cells. most enveloped viruses enter cells through cellular endocytosis [ , ] . the endocytic pathways utilized by viruses vary, including clathrin-mediated endocytosis (cme), caveolaemediated endocytosis, lipid raft-mediated endocytosis, and macropinocytosis, among others. cme is the most classical and well-known endocytic pathway utilized by viruses. after binding to cell surface receptors, the virus is packaged by clathrin-coated pits (ccps) and transported to clathrin-coated vesicles (ccvs), in which virus particles as the "cargo" will be transported to the early endosomes [ , ] . caveolae is a plasma-specific invagination structure with a diameter of - nm. when viral particles interact with receptors, caveolae coated with caveolin- invaginates and pinches off plasma membrane, then the caveolae vesicles mature into caveosomes and deliver "cargoes" to early endosomes [ , ] . lipid raft are plasma membrane microdomains enriched in sphingolipids and cholesterol that participate in the lateral organization of the cell surface. raft-mediated endocytosis is the process of internalization of ligands and receptors by these domains [ ] . the mechanisms of some coronaviruses entry into cells have already been studied, such as severe acute respiratory syndrome coronavirus (sars-cov), murine hepatitis virus (mhv), and human coronavirus (hcovs). entry of sars-cov into hepg and cos cells is clathrindependent while entry into vero e cells is clathrin-and caveolae-independent [ , ] , but the lipid raft plays an important role in the process [ ] . mhv entry into cells needs clathrin [ ] [ ] [ ] , the same as hcov-nl [ ] . for hcov- e, caveolae-mediated endocytosis is utilized to enter human fibroblast cells [ ] . sars-cov and mhv-cov can induce continuous micropinocytosis, but this occurs in the later phase during infection and is not associated with virus entry [ ] . coronaviruses enter host cells via various endocytic pathways after viral spike glycoprotein (s) interacts with receptors and then initiates the endocytic process. internalized viruses are trafficked like cargoes to membrane fusion sites through specific transport routes. different covs have varying fusion sites [ ] . the fusion site of the middle east respiratory syndrome coronavirus (mers-cov) takes place in the early endosome, while mhv and the feline infectious peritonitis virus (fipv) are transported to the lysosome to fuse. although there have been many studies on the invasion mechanism of cov, the invasion strategy of pedv has not yet been fully elucidated. in , park et al. [ ] revealed that pedv entry followed clathrin-mediated endocytosis and was dependent on a low ph for successful entry into cells. in their research, one pedv strain was studied in vero cells in the presence of trypsin and only the chemical inhibitors and confocal method were used to reveal the pedv entry. however, there are still important questions to address. considering that covs take advantage of different pathways to enter cells, whether different subtypes of pedv invade cells by different ways and whether pedv enter different types of cells through different ways remains to be determined. to concretely clarify the entry and transportation routes of pedv, we used vero and ipec-j cells as models for pedv entry and chose cv -like strain gds and highly pathogenic variant strain gds to compare the invasion strategies of different pedv subtypes. our results will advance the understanding of the pathogenesis and immune evasion of pedv. vero cells were cultured in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs, gibco) and antibiotics ( u/ml penicillin and μg/ml streptomycin). ipec-j cells were grown in dmem/nutrient mixture f- (dmem/f ) supplemented with % fbs and antibiotics. the pedv strains used in this study were cv -like strain gds (gi subtype, genbank id: mh . ) and highly pathogenic variant strain gds (gii subtype, genbank id: km . ). exogenous trypsin ( μg/ml) was added to proliferate pedv strains in vero cells and μg/ ml trypsin was added in ipec-j cells. sbti (soy bean trypsin inhibitor type i; sigma no. t ). the endocytic inhibitors used included dynasore (sigma-aldrich, no. ), chlorpromazine (cpz, sigma-aldrich, no. c ), methyl-β-cyclodextrin (mβcd, sigma-aldrich, no. c ), nystatin (sigma-aldrich, no. ), ammonium chloride (nh cl, sigma-aldrich, no. a ), and bafilomycin a (baf a , sigma-aldrich, no. ). antibodies against clathrin heavy chain, caveolin , eea , rab , and lamp coupled with secondary goat anti-rabbit alexa fluor and goat anti-mouse alexa fluor were purchased from abcam. the mouse anti-pedv-s monoclonal antibody [ ] and anti-pedv-n polyclonal antibody (prepared in our laboratory) were used in the immunofluorescence analysis and western blotting analysis, respectively. the overexpression plasmids of wild-type and mutant dynamin ii (gfp-dyn-wt and gfp-dyn-m), eps (gfp-eps -wt and gfp-eps -m), and caveolin- (gfp-cav-wt and gfp-cav-m) were provided by prof. mark mcniven, mayo center for biomedical discovery (rochester, mn, usa). to investigate the trypsin dependency of pedv strains, vero cells were seeded in -well plates until confluence. after washed with pbs, cells were infected with pedv strains at a multiplicity of infection (moi) of . with or without trypsin ( μg/ml) or with trypsin and μg/ml sbti for h before the quantification of the viruses by qrt-pcr. to test the dynamics of pedv internalization, vero or ipec-j cells were seeded in -well plates until confluence. the cells were pre-chilled for min and inoculated with pedv at a moi of . at °c for h for virus binding. the cells were washed three times with ice-cold pbs to remove unbounded viruses and immediately warmed to °c to initiate internalization. after incubation for the indicated time intervals, the cells were treated with proteinase k ( mg/ml) at °c for min and then washed with pbs to inactivate and remove the non-internalized pedv particles. the control cells were then washed with pbs. the cells were collected and subjected to qrt-pcr analysis [ ] . to test the effect of inhibitors on pedv internalization, it was necessary to evaluate the cytotoxicity of cell inhibitors. the cells were seeded in -well plates at a density of × cell/well, grown for h, and treated with endocytic inhibitors at the indicated concentration for h. then μl of cck- solution was added to each well and incubated at °c for h. an absorbance of nm was measured. the experiments were repeated three times independently. the concentration of each used inhibitor did not cause significant cytotoxicity to the cell viability. to test the effect of inhibitors on pedv internalization, the cells were pre-treated with different concentrations of drugs for h and then infected with gds or gds strains at moi = in the presence of drugs for h. after washing with citrate buffer (ph . ) [ ] and pbs, the cells were incubated with medium containing trypsin for h or h at °c and collected for qrt-pcr and western blotting analysis, respectively. the expression of pedv n protein was detected by qrt-pcr and western blotting with gapdh as the reference. total rna was extracted using trizol (invitrogen) according to the manufacturer's instruction and cdna was synthesized with a revertra ace qpcr rt master mix with gdna remover kit (toyobo, osaka, japan). qpcr reaction was performed using a sybr premix ex taq ii kit (takara, tokyo, japan) using a light cycler real-time pcr system (roche diagnostics, indianapolis, in, usa). for the western blotting analysis [ ] , the cells were washed with pbs and lysed in ripa lysis buffer on ice for min. after sds-page electrophoresis, proteins were transferred onto polyvinylidene fluoride (pvdf) membrane via the semidry method and immunoblotted with the corresponding antibodies. transfection of vero cells and ipec-j cells with the overexpression plasmids of wild-type and mutant dynamin ii (gfp-dyn-wt and gfp-dyn-m), eps (gfp-eps -wt and gfp-eps -m), and caveolin- (gfp-cav-wt and gfp-cav-m) were performed using lipofectamine (invitrogen) transfection reagents according to the manufacturer's protocol. the cells were seeded in -well plates until % confluence. h after transfection, the cells were infected with pedv at moi = for h. virus was moved with citrate buffer and pbs and replaced with fresh medium containing trypsin, and virus internalization was evaluated by confocal fluorescence microscope. for the rna interference assay, sirnas against dynamin ii (sidyn, sus scrofa: ′-cac ctc atg atc aat aac a- ′, chlorocebus sabaeus ′-cct aca tca aca cga acc a- ′), clathrin heavy chain (sichc, sus scrofa ′-ccc ata cca tga ctg atg a- ′, chlorocebus sabaeus ′-gat gaa cct tat gca tgc a- ′), eps (sieps , sus scrofa ′-cct gtg gat att ctt gga a- ′, chlorocebus sabaeus ′-ccc aga aac agc aag tac a- ′), and caveolin- (sicav, sus scrofa ′-caa cat gca gaa aga aat a- ′, chlorocebus sabaeus ′-cct tca ctg tga cga agt a- ′) were designed and synthesized based on the corresponding full-length mrna sequences of sus scrofa and chlorocebus sabaeus, sir-nas against rab a (sirab , ′-gat ggt gga tga cag act a- ′), and vps (sivps , ′-gct tca aga gag act act a- ′) were designed and synthesized based on the corresponding mrna homologous sequences of sus scrofa and chlorocebus sabaeus. the control sirna (sicontrol) was designed and synthesized irrelevantly to all the known genes of sus scrofa and chlorocebus sabaeus genome, respectively, by ribobio (guangzhou, china). the cells were seeded in -well plates until % confluence. to ensure transfection efficiency, a second transfection was carried out at h after the first transfection. at h post-first transfection, the cells were infected with pedv at moi = for h. virus was moved with citrate buffer and pbs and replaced with fresh medium containing trypsin, and virus internalization was evaluated by qrt-pcr and western blotting at hpi and hpi, respectively. alexa- labeled transferrin (trf ) or alexa- labeled cholera toxin b subunit (ctb) were diluted at : and mixed with pedv at moi = . the cells were washed three times with pbs and added to the mixture of pedv and trf or ctb at °c for h and then incubated at °c for min for internalization. after washing with pbs, the cells were fixed, permeabilized, blocked, incubated with mouse anti-pedv-s monoclonal antibody, incubated with alexa -conjugated goat anti-mouse igg (h + l), stained with dapi, and analyzed using a confocal fluorescence microscope. light exposure was avoided throughout this experiment. cells cultured in glass-bottom dishes for h were washed with ice-cold pbs and incubated with pedv at °c for h. cold viruses were replaced with pre-warmed medium, and the cells were immediately shifted to °c. at specific time points, the cells were fixed in % paraformaldehyde at rt for min after washing three times with pbs. permeabilization was carried with . % triton x- at rt for min. after washing with pbs, the cells were blocked with % bsa in pbst at rt for min to block unspecific binding sites. the specific primary antibodies against chc, eea , caveolin- , rab , lamp , and anti-pedv-s antibody were used to probe the cells at °c overnight. the cells were incubated with secondary antibodies (goat anti-rabbit igg antibody conjugated to alexa fluor and goat anti-mouse igg antibody conjugated to alexa fluor ) at °c for h. fluorescent images were acquired using the light-scanning module of a leica tcs sp sted × confocal microscope. the cells ( × ) were incubated or not incubated with pedv at °c for h, washed three times with ice-cold pbs, and lysed in ml tne buffer ( mm tris, mm nacl, mm edta, and ph . ) containing % triton x- and % phenylmethanesulfonyl fluoride (pmsf) on ice for min. the homogenized cell lysates were centrifuged at °c for min at g and the supernatant was mixed with isometric ml containing % sucrose in tne buffer. the lysates-sucrose mixture was placed at the bottom of ultracentrifugal tubes and overlaid with ml % and ml % sucrose in tne buffer. the cell lysates were ultracentrifuged at °c for h at g in a sw rotor (beckman). after centrifugation, twelve ml fractions were collected from the top to the bottom of the tubes. the fractions were concentrated with % peg at °c overnight, and the pellets were resuspended in μl of tne buffer after centrifuging at °c for min at g. the localizations of lipid raft-associated protein caveolin- and pedv n protein were analyzed by western blotting. all the graphs were created with graphpad prism software. all the data are presented as the means ± standard deviations (sds) from at least three independent experiments. significance was estimated using one-way anova with multiple comparisons to control. p values less than . were defined as the threshold for statistical significance. p values between . and . were marked with one asterisk, p values between . and . were marked with two asterisks, p values between . and . were marked with three asterisks, and p values less than . were marked with four asterisks. coronavirus entry is inextricably linked with proteolytic processing of the s protein. in most cases, pedv is trypsin dependent. thus, we investigated the trypsin dependency of both strains used in our research. as shown in figure a , gds strain needed trypsin while gds strain is trypsin independent. so, we added trypsin in the following assays to explore the invasion mechanism of pedv. the dynamics of viruses invading different kinds of cells vary, and there may be differences among various subtypes of the same virus. thus, it is necessary to know the entry dynamics of pedv before studying the endocytic pathways. the cells were incubated with pedv at moi = . at °c for adsorption and shifted to °c to initiate internalization. the adsorbed but not internalized virions were removed with proteinase k, and the pedv invasion rates at different time points were detected using qrt-pcr. the invasion kinetics ( figure ) showed that most of the pedv particles were detached from the cells by proteinase k at the beginning of invasion. after min in the vero cells ( figure b ) and min in the ipec-j cells ( figure c ), nearly maximum proportions of viral particles completed the internalization. approximately % of the pedv particles entered the vero cells, while only % entered the ipec-j cells. notably, the gds strain demonstrated less efficient invasion than the gds strain, but there was no significant difference. dynamin ii plays an essential role in cellular membrane fusion during vesicle formation due to its gtpase activity, and it is necessary for clathrin-and caveolae-mediated endocytosis [ , ] . thus, we explored the essentiality of dynamin ii in pedv entry using specific chemical inhibitors, overexpression of domain-negative mutants of dynamin ii, and sirna interference. dynasore [ ] , a cell-permeable non-competitive inhibitor of dynamin ii, was used to pre-treat cells at different concentrations to analyze the effect on pedv entry. the cytotoxicity test showed that μm of dynasore had no effect on the viability of the vero and ipec-j cells (additional file ). cells were pre-treated with μm and μm dynasore for h before pedv infection. dmso was used as a negative control. the effect of dynasore on pedv entry was quantified by qrt-pcr at hpi. pedv invasion was significantly inhibited by dynasore. at a concentration of μm, the invasion rates of the gds and gds strains into the vero cells were approximately % and %, and the invasion rates into the ipec-j cells were % and %, respectively ( figure a) . a comparison of the invasion rates of the gds and gds strains indicated that the gds strain was more sensitive to dynasore than the gds strain when invading the ipec-j cells but there was no significant difference between them when invading the vero cells. many studies used the overexpression of dominant negative mutants to explore the role of dynamin ii in virus entry [ , ] . mutation of dynamin ii from k to a can inhibit gtpase activity and reduce endocytosis [ ] . cells were transfected with wild-type and mutant types of dynamin ii respectively and infected with gds and gds strains at h after transfection. the confocal results showed that the vero and ipec-j cells overexpressing wild-type dynamin ii (gfp-dyn-wt) were infected with pedv while the cells overexpressing mutant dynamin ii (gfp-dyn-m) were barely infected ( figure b ). sirna interference was also used to identify the importance of dynamin ii on virus entry [ , , ] . sus scrofa and chlorocebus sabaeus sirnas of dynamin ii (sidyn) were designed and synthesized. the interference efficiency of sirna on the dynamin ii expression in the vero and ipec-j cells was obvious at both the mrna and protein levels (additional file ). cells were infected with pedv after transfection twice and the internalized virions were quantified at hpi and hpi by qrt-pcr and western blotting assay, respectively. the qrt-pcr results ( figure c ) showed that the knockdown of dynamin ii expression reduced the pedv internalization. the internalization rates of the gds and gds strains into vero cells were approximately % and %, the internalization rates into the ipec-j cells were approximately % and %, respectively, but there was no significant difference between the gds and gds strains in the two cells ( figure c ). the same results were confirmed by western blotting ( figure d ). taken together, the results suggested that pedv entry relies on dynamin ii. clathrin-mediated endocytosis is the most commonly used and classical endocytic pathway for virus entry. to identify whether pedv utilized cme to enter cells, we co-inoculated the vero and ipec-j cells with pedv and trf, which is the most typical biomolecule that uses cme to enter cells [ ] . the co-inoculation results figure trypsin-dependency and kinetics of pedv entry into cells. a vero cells were seeded in -well plates until confluence. cells were washed with pbs and infected with pedv strains (moi = . ) without trypsin or in the presence of trypsin ( μg/ml) or trypsin and μg/ml sbti. cells were collected for qrt-pcr at hpi. b, c vero cells (b) and ipec-j cells (c) were incubated with pedv gds and gds strains, respectively, at °c for h and shifted to °c immediately to initiate internalization. at , , , , , , , , and min after incubation, the cells were treated with proteinase k ( mg/ml) at °c for min to inactivate the non-internalized virions. the control cells were washed with pbs. the invasion rates were calculated by qrt-pcr analysis. ****p < . . dynamin ii involved in pedv entry. a cells were pre-treated with μm and μm dynasore at °c for h, respectively, and incubated with gds or gds strain for h. dmso was used as a negative control. the cells were collected at hpi for qrt-pcr assay to test the invasion efficiency of pedv. b vero and ipec-j cells were transfected with gfp-dyn-wt and gfp-dyn-m, respectively, and infected with pedv strains at h after transfection. the cells were fixed at hpi and stained for confocal analysis. c, d vero and ipec-j cells were transfected with sidyn twice and infected with pedv strains at h after the second transfection. the invasion rates of pedv into the cells were detected at hpi and hpi for qrt-pcr and western blotting analysis, respectively. ctrl means control. scale bars indicate μm. ** . < p < . ; *** . < p < . ; ****p < . . showed that pedv co-located with trf in the two types of cells ( figure a ), which means that pedv might utilize clathrin-mediated endocytosis to enter cells. to prove this conjecture, we used specific chemical inhibition, the overexpression of domain negative mutants of eps , the knockdown expression of chc and eps by sirna, and the location of pedv in the cells to estimate the role of clathrin-mediated endocytosis in pedv entry. cpz is a specific chemical inhibitor used to block the cme pathway by preventing the assembly of ccps at the plasma membrane [ ] . the cytotoxicity test showed that μm cpz have no effect on the viability of the vero cells and μm cpz have no effect on the viability of the ipec-j cells (additional file ). vero and ipec-j cells were treated with cpz at different concentrations for h and then infected with pedv. the internalization rates of pedv after cpz treatment were quantified by qrt-pcr and western blotting at hpi and hpi, respectively. the qrt-pcr results (figure b ) showed that pedv invasion was significantly inhibited by cpz. the invasion rates of the gds and gds strains at the highest drug concentrations were nearly % and % in the vero cells and % and % in the ipec-j cells, respectively. notably, there were no significant differences between the gds and gds strains in the vero cells but the gds strain was more sensitive than the gds strain in the ipec-j cells, reflecting the gds strain's significantly decreased invasion rates ( figure b ). the same results were also observed by western blotting ( figure c ) and ifa assay (additional files , ) . the role of cme in endocytosis was also identified by the overexpression of gfp-tagged dominant negative mutants of eps [ ] . eps is a critical component of ccps by interacting with adaptor protein (ap- ), a major clathrin adaptor complex [ ] . cells transfected with wild-type (gfp-eps -wt) and mutant eps (gfp-eps -m) were infected with pedv strains at h after transfection. the confocal results of the pedv invasion showed that the cells overexpressing wild-type eps were infected with pedv while few infections were observed in the overexpressed gfp-eps -m cells ( figure d ). sirna was also used to explore the role of cme in pedv entry by interfering with the expression of clathrin heavy chain (chc) and eps . chc and clathrin light chain form a triskelion shape, which is a key component for regulating the formation and disassembly of the clathrin lattice [ ] . cells were infected with pedv strains after transfection twice, and the invasion rates of the viruses were assessed using qrt-pcr and western blotting assay at hpi and hpi, respectively. the quantitative experiments showed that knockdown of the expression of chc significantly reduced the invasion rates of pedv. the invasion rates of the gds and gds strains were % and % in the vero cells and % and % in the ipec-j cells, respectively, and there was no significant difference between the gds and gds strains ( figure e ). knockdown of the expression of eps also significantly reduced the invasion rates of pedv. the invasion rates of the gds and gds strains were % and % in the vero cells and % and % in the ipec-j cells, respectively, and there was no significant difference between the gds and gds strains ( figure g ). the significant inhibition of sichc and sieps on pedv entry was also observed by western blotting assay (figures f, h) . to estimate whether pedv directly entered the cells through cme, we analyzed the localization of pedv and chc in the vero and ipec-j cells, respectively. pre-cooled cells were incubated with pedv at °c for h for adsorption and shifted to °c for internalization. five min later, the cells were washed and fixed for observation using an ultrahigh-resolution laser confocal microscope. the confocal results showed that pedv particles co-located with chc protein in the vero and ipec-j cells ( figure i ), but some virions were not co-localized with chc. the results indicated that pedv can enter cells through the cme pathway, but cme may not be the only pathway utilized by pedv. cholesterol, an important component of cell membranes, embeds phospholipid bilayers and plays a crucial role in the fluidity of cell membranes [ ] . many studies showed that most enveloped virus relied on cholesterol to invade cells [ , ] . if a virus invades cells, depending on the presence of cholesterol, it will be sensitive to cholesterol extractants. mβcd can eliminate cholesterol on the plasma membrane of cells [ ] . nystatin can bind to the cholesterol-enriched regions of cell membrane and then decompose cholesterol and impair cholesterol synthesis [ ] . the cytotoxicity test showed that the maximum tolerance concentrations of vero and ipec-j cells to mβcd were mm and . mm, respectively, and the maximum tolerance concentrations to nystatin were μm and μm, respectively (additional file ). cells were pre-treated with different concentrations of mβcd and nystatin for h, then infected with pedv. the effects of drugs on pedv entry were estimated by qrt-pcr and western blotting at hpi and hpi, respectively. mβcd showed a significant inhibition of pedv entry. the internalization rates of the gds and gds strains after mβcd treatment were approximately % and % in the vero cells and approximately % and % in the ipec-j cells. there were no significant differences between the gds and gds strains in the mβcd-treated vero figure pedv entry relies on the cme pathway. a vero cells and ipec-j cells were incubated with mixture of alexa- labeled trf (red) and pedv (green) at °c for h, and then shifted to °c for min. the cells were fixed and stained for pedv using monoclonal antibody against pedv s protein. the cellular localizations of trf and pedv were observed with a confocal fluorescence microscope. light exposure was avoided throughout this process. b, c. the vero cells were pre-treated with μm and μm of cpz, and the ipec-j cells were pre-treated with μm and μm of cpz, respectively, at °c for h and incubated with gds or gds strains for h. double-distilled water was used as a negative control. the cells were collected at hpi and hpi for qrt-pcr and western blotting assay, respectively, to test the invasion efficiency of pedv. d the vero cells (left) and ipec-j cells (right) were transfected with gfp-eps -wt and gfp-eps -m, respectively, and infected with pedv strains at h after transfection. the cells were fixed at hpi and stained for confocal analysis. e-h the vero cells and ipec-j cells were transfected with sichc and sieps and infected with pedv strains at h after the second transfection. the cells were collected at hpi and hpi for qrt-pcr and western blotting analysis, respectively. i the cells were pre-cooled at °c for min, incubated with pedv strains at °c for h, shifted to °c for min to initiate internalization, and washed for three times to remove un-internalized viral particles. the cells were fixed and stained with anti-pedv-s (red) and anti-chc (green) primary antibodies. ctrl means control. scale bars indicate μm in a, μm in d, and μm in i. ** . < p < . ; *** . < p < . ; ****p < . . cells but in the ipec-j cells, the gds strain was more sensitive to mβcd ( figure a ). the results were confirmed by western blotting ( figure b ) and ifa assay (additional files and ). pedv entry was significantly inhibited by nystatin treatment. the internalization rates of the gds and gds strains after nystatin treatment were approximately % and % in the vero cells and approximately % and % in the ipec-j cells. the gds strain was more sensitive to nystatin than the gds strain in both the vero and ipec-j cells ( figure c ). the same results were confirmed by western blotting ( figure d ) and ifa assay (additional files and ). to further evaluate the importance of cholesterol, cells pre-treated with mβcd were supplemented with exogenous cholesterol and then infected with pedv, and the changes in the viral invasion rates were quantified by qrt-pcr at hpi. the results showed that adding exogenous cholesterol could significantly increase the invasion rate of pedv. the average invasion rate of the gds and gds strains increased from % to over % in the vero cells and from % and % to approximately % in the ipec-j cells ( figure e ). the results indicated that pedv invading and entering cells depended on cholesterol but the two pedv subtypes showed different degrees of dependence on cholesterol when entering the vero and ipec-j cells. as both caveolae and lipid raft are rich in cholesterol, they are sensitive to cholesterol inhibitors [ , ] . to identify whether caveolae-mediated endocytosis was involved in pedv entry, cells were co-inoculated with pedv and ctb, which entered the cells after interactions with specific receptors [ ] . the localization results showed that pedv co-localized with ctb in vero and ipec-j cells ( figure a ), which means pedv may utilize caveolae-mediated endocytosis to enter cells. as the caveolae is mainly coated with caveolin- [ ] , knocking down the expression and separating the interaction factors with caveolin- blocked the caveolae-mediated endocytic pathway. overexpression of the domain-negative mutant of caveolin- [ ] blocked its interaction with the interaction factors. cells were transfected with wild-type caveolin- (gfp-cav-wt) and mutant caveolin- (gfp-cav-m), then infected with pedv at h after transfection, and fixed for confocal observation at hpi. the results showed that pedv infected gfp-cav-wt-overexpressing cells but barely infected gfp-cav-m-overexpressing vero or ipec-j cells ( figure b ). sirnas (sicav) were designed and synthetized to knockdown caveolin- expression. cells were transfected with sicav twice and then infected with pedv. after the second transfection, the invasion rates of pedv were measured by qrt-pcr and western blotting at hpi and hpi, respectively. the qrt-pcr results showed that the knockdown of caveolin- expression reduced the internalization of pedv. the inhibition rates in the gds and gds strains were % and % in the vero cells and % and % in the ipec-j cells, respectively (figure c) . compared with the gds strain, the gds strain showed a higher degree of reduction in the invasion rate in the vero cells but there was no significant difference in the ipec-j cells ( figure c ). the same results were confirmed by western blotting assay ( figure d ). to identify the role of caveolae in pedv entry, we investigated the cellular localization of pedv with caveolin- . pre-cooled cells were incubated with pedv at °c and then shifted to °c for internalization. the cells were then washed and fixed for min for observation with an ultrahigh-resolution laser confocal microscope. the cellular localization results showed that pedv was colocated with caveolin- in the vero and ipec-j cells ( figure e ). pedv can enter cells through the caveolaemediated pathway. if pedv can enter cells through the lipid raft pathway, the viral components should be contained in lipid raft enrichment layer after isolated by sucrose density gradient centrifugation [ ] . after incubation with pedv, the cells were lysed and subjected to sucrose gradient centrifugation. the products were extracted from the top down and a total of fractions were obtained for western blotting analysis. caveolin- was used as the protein marker representing the lipid raft layer [ ] . the results showed that pedv could be detected in the upper lipid raft enrichment layer. almost all the virions were concentrated in the lipid raft enrichment layer in the vero cells ( figure a ) and virions were detected in both the upper and lower components in the ipec-j cells ( figure b ). the differences between the gds and gds strains were the proportion of virions in the upper and lower components in the ipec-j cells. the gds particles were mainly present in the upper layer while amounts of gds particles were present in the lower layer (figure b) . the results indicated that pedv utilized lipid rafts to enter cells. viruses that enter cells via endocytosis are usually trafficked by endocytic vesicles to early endosomes for sorting and are transported to late endosomes or fused with early endosomes [ ] . if viruses do not fuse in the early endosomes and release genomes into the cytoplasm, they will enter the late endosomes with the further acidification and maturation of the early endosomes. similarly, figure pedv entry relies on cholesterol. a, b vero cells and ipec-j cells were pre-treated with . mm and mm and mm and . mm mβcd, respectively, at °c for h and incubated with gds or gds strains for h. double-distilled water was used as a negative control. the cells were collected at hpi and hpi for qrt-pcr and western blotting assay, respectively, to test the invasion efficiency of pedv. c, d the vero cells and ipec-j cells were pre-treated with μm and μm and μm and μm of nystatin, respectively, at °c for h and incubated with gds or gds strains for h. dmso was used as a negative control. the cells were collected at hpi and hpi for qrt-pcr and western blotting assay, respectively, to test the invasion efficiency of pedv. e the vero cells and ipec-j cells were pre-treated with different concentrations of mβcd at °c for h, supplemented with μg/ml of soluble cholesterol at °c for h, and infected with pedv strains for h. the cells were collected at hpi for qrt-pcr assay to test the invasion efficiency of pedv. *p < . ; ** . < p < . ; *** . < p < . ; ****p < . . a vero cells and ipec-j cells were incubated with a mixture of alexa- labeled ctb (red) and pedv (green) at °c for h, and then shifted to °c for min. the cells were fixed and stained for pedv using monoclonal antibody against s protein. the cellular localizations of ctb and pedv were observed with a confocal fluorescence microscope. light exposure was avoided throughout this process. b vero cells (up) and ipec-j cells (down) were transfected with wild-type caveolin- (gfp-cav-wt) and domain negative mutant of caveolin- (gfp-cav-m), respectively, and infected them with pedv strains at h after transfection. the cells were fixed at hpi and stained for confocal analysis. c, d the vero cells and ipec-j cells were transfected with sicav twice and infected with pedv strains at h after the second transfection. the cells were collected at hpi and hpi for qrt-pcr and western blotting analysis, respectively. ctrl means control. e cells were pre-cooled at °c for min, incubated with pedv strains at °c for h, shifted to °c to initiate internalization for min, and washed for three times to remove viral particles that were not internalized. the cells were fixed and stained with anti-pedv-s (red) and anti-caveolin- (green) primary antibodies. scale bars indicate μm in a, μm in b, and μm in e. *p < . ; ** . < p < . ; *** . < p < . ; ****p < . . if the ph environment in the late endosomes does not meet the requirements for conformational changes of viral glycoproteins to cause membrane fusion, viruses will enter the lysosomes with the transportation of the late endosomes and finally achieve membrane fusion. viruses require acidic ph to traffic between endosomes. therefore, it is necessary to clarify whether pedv relies on a low ph environment. nh cl is an inhibitor of endosome acidification [ ] . baf a is a v-atpase inhibitor that can block the traffic of endocytic cargoes from early endosomes to late endosomes and inhibit the stability of low ph environments in the lysosome lumen [ ] . cells were pre-treated with different concentrations of nh cl and baf a (additional file ) and infected with pedv (additional file ). the invasion rates of pedv were measured by qrt-pcr and western blotting at hpi and hpi, respectively. the results showed that cells pretreated with nh cl significantly inhibited pedv entry. the invasion rates in the gds and gds strains were approximately % and % in the vero cells and % and % in the ipec-j cells ( figure a ). this significant inhibition was also confirmed by western blotting (figure b ) and ifa assay (additional files and ). baf a can significantly inhibit pedv entry. the invasion rates in the gds and gds strains were approximately % and % in the vero cells and % and % in the ipec-j cells ( figure c ). western blotting ( figure d ) and ifa assay (additional files , ) also confirmed the inhibition of baf a . the results proved that pedv entry requires low ph. early endosomes mature into late endosomes by increasing intraluminal acidity through proton pump activity. late endosomes can become larger vesicles by fusing with the same type of endosomes, so they mostly exist in the form of a multivesicular body (mvb). late endosomes release rab , incorporate rab , and prepare to fuse with lysosomes [ ] . to explore whether pedv particles are trafficked after internalization, we interfered with the expression of rab [ ] involved in late endosomes and vps [ ] involved in late endosometo-lysosome maturation and identified whether viral particles co-located with early endosomes, late endosomes and lysosomes. knockdown of the expression of rab (sirab ) can significantly inhibit the invasion efficiency of pedv. the invasion rates of the gds and gds strains were % and % in the vero cells and % and % in the ipec-j cells ( figure a) . similarly, the knockdown of the expression of vps (sivps ) also significantly inhibited the invasion efficiency of pedv. the invasion rates of the gds and gds strains were % and % in the vero cells and % and % in the ipec-j cells ( figure b ). for cellular location by pedv observation, pre-cooled cells were incubated with pedv at °c and then shifted to °c for internalization. the cells were washed and fixed at different time points after shift for observation using an ultrahigh-resolution laser confocal microscope. cellular localization assays showed that internalized pedv could co-locate with eea , the early endosome protein marker, in the vero were incubated or not with pedv at °c for h and then lysed in tne buffer containing % triton x- and % pmsf on ice for min. after mixing with isometric % sucrose, the homogenized cell lysates were subjected to ultracentrifugation after being overlaid with % and % sucrose. after centrifugation, a total of fractions were collected from the top to the bottom of the tubes. the localizations of the lipid raft-associated protein caveolin- and pedv n protein were analyzed by western blotting after being concentrated with % peg . and ipec-j cells min after endocytosis ( figure c ) and could co-locate with rab , the late endosome protein marker, min after endocytosis ( figure d ), while the late endosomes were mostly in the form of mvb. colocalization of pedv with lamp (lysosomal associated membrane protein ), an important lysosome membrane component, was also observed min after endocytosis in the two types of cells ( figure e ). the results demonstrated that pedv was trafficked to the lysosomes after entering the cells through endocytosis, and there were no differences between the two pedv genotypes and cells. since highly pathogenic variant strains emerged in , pedv has attracted global attention. many studies have reported that vaccines based on cv or cv -like strains have low protection efficiency against re-emerging variant strains [ , , [ ] [ ] [ ] . genotyping showed that pedv strains can be sorted into two genotypes, gi subtypes (classical) and gii subtypes (variant). the nucleotide sequence of s subunit of s protein is - % similar between gi and gii, which shows high variability [ , ] . as the main antigen of pedv, the s protein incubated with gds or gds strains for h. double-distilled water was used as a negative control. the cells were collected hpi and hpi for qrt-pcr and western blotting assay, respectively, to test the invasion efficiency of pedv. c, d the vero cells and ipec-j cells were pre-treated with nm and nm baf a at °c for h and then incubated with gds or gds strains for h. dmso was used as a negative control. the cells were collected hpi and hpi for qrt-pcr and western blotting assay, respectively, to test the invasion efficiency of pedv. ** . < p < . ; *** . < p < . ; ****p < . . vero cells and ipec-j cells were transfected with sirab and sivps twice, respectively, and then infected with pedv strains at h after the second transfection. the cells were collected at hpi for qrt-pcr analysis. ctrl means control. c-e the vero cells and ipec-j cells were pre-cooled at °c for min, incubated with pedv strains at °c for h, shifted to °c to initiate internalization. the non-internalized viral particles were removed by washing. min after shifting, the cells were fixed and stained with anti-pedv-s (red) and anti-eea (green) primary antibodies (c). min after shifting, the cells were fixed and stained with anti-pedv-s (red) and anti-rab (green) primary antibodies (d). min after shifting, the cells were fixed and stained with anti-pedv-s (red) and anti-lamp (green) primary antibodies (e). scale bars indicate μm in c-e. *p < . ; ** . < p < . ; *** . < p < . ; ****p < . . plays an important role in inducing immune responses and viral entry. mutation of the s gene may lead to different mechanisms of virus invasion, which may help elucidate the pathogenesis and immune evasion of pedv. as sars-cov utilizes varying endocytic routes to invade different cells [ , ] , we wondered whether pedvs can enter cells through different pathways. burkard et al. clarified that coronavirus entered cells through the endosome/lysosome pathway and was proteolytic dependent. the furin cleavage site just upstream of the fusion peptide (fp) of the s protein was the key to determining the fusion site of the viral membrane [ ] . while the s protein of pedv does not have the furin cleavage site, a conserved arginine just upstream of the putative fp as the potential cleavage site can be cleaved by trypsin [ ] . whether the theory mentioned above is applicable to pedv thus needs further study. here, we explored the gi and gii subtype pathways of pedv entry into vero and ipec-j cells, respectively, and the transportation route after internalization. our results showed that two the subtypes of pedv utilized clathrin-, caveolae-, and lipid raft-mediated endocytosis to enter the vero and ipec-j cells, but the utilization efficiency of each endocytic pathway varied depending on the different genotypes and types of cells. to describe the dynamic curve of pedv entry, the appropriate viral scavenger is extremely important to remove virus particles effectively adsorbed on the cell surface. we compared the effects of citrate buffer (ph . ) with proteinase k ( mg/ml), and the latter exhibited a stronger capacity to remove viruses. the results of dynamic invasion showed that the virus invaded vero cells % within min, while the invasion efficiency of the ipec-j cells was only approximately %. although ipec-j cells are considered the host cells of pedv, the cell lines cultured in vitro lost their polar growth state in vivo [ ] , which possibly affects the viral recognition and reduces the infection efficiency of the virus. gds showed a lower invasion rate than gds , but there was no significant difference. dynamin ii, a gtpase, plays an important role in endocytosis by pinching the endocytic vesicles off the plasma and is necessary in clathrin-and caveolae-mediated endocytosis. we found that pedv entry is dynamin iidependent, although the sensitivity of the gds and gds strains to dynasore varied. considering the low specificity of chemistry inhibitor, dominant negative mutant and sirna-mediated knockdown of dynamin ii were carried out to evaluate dynamin ii for pedv infection. both the gds and gds strains were inhibited by sidynamin ii with no significant difference. clathrin-mediated endocytosis is a classical and commonly used pathway for most enveloped viruses. many coronaviruses use cme to enter cells, such as sars-cov entry into hepg cells and cos cells and phev entry into neuro- a cells [ , ] . to ascertain whether pedv utilized cme to enter cells, the chemistry inhibitor cpz was used to prevent clathrin assembly and further block cme. clathrin is composed of light chain (clc) and heavy chain (chc) which form clathrin lattices under the interaction of ap- and eps . both cpz pre-treatment and sirna-mediated knockdown of chc and eps can significantly reduce the invasion rate of pedv into cells, with no significant difference between the gds and gds strains. dominant negative mutants can provide a more specific method to study endocytic pathways by separating the prototype protein from their interaction regulatory factors. in this study, we also showed that cells overexpressing dominant negative mutant of eps were barely infected with pedv but cells overexpressing wild-type eps were infected as normal. however, the inhibition of endocytosis by overexpressing dominant negative mutants may be compensated through other clathrin-independent endocytic pathways. when viruses enter cells through cme, they are carried by clathrincoated vesicles. co-localization of viral particles and chc indicated that pedv entry relies on cme. although caveolae and lipid rafts have the same components, such as caveolin- , gm , and cholesterol, they are two completely different endocytic pathways. before investigating whether pedv can use these two pathways to enter cells, we first examined whether pedv invasion depends on cholesterol. the cholesterol inhibitors nystatin and mβcd had significant inhibitory effects on pedv entry. nystatin had higher inhibitory effects on gds entry than gds . the inhibitory effects of mβcd on vero cells were similar for gds and gds strains. mβcd effects were lower on gds than gds in ipec-j cells. we hypothesized that gds cell invasion mainly depended on cholesterol on the cell surface, while gds depended on the presence of cholesterol on the cell surface and cholesterol synthesis. exogenous cholesterol supplementation also confirmed the importance of cholesterol in pedv entry. endocytic vesicles formed in the caveolae-mediated pathway were coated with caveolin- , which plays a critical role in the process. dominant negative mutant, rna interference, and the cellular co-localization of caveolin- with viral particles provided further evidence that pedv entry needed caveolin- . collectively, both subtypes of pedv entered the vero and ipec-j cells through caveolae-mediated endocytosis. however, park et al. [ ] showed that pedv entry was independent of caveolae-coated pit assembly by treating vero cells with nystatin. the different results may be explained by different operational details. firstly, nystatin was used before and during the incubation of pedv in this study, while only before incubation in the research of park et al. the concentrations of nystatin used in the two studies were different. the highest concentration of nystatin used in park's research was μm [ ] , while the highest concentration we used was μm in vero cells. when the concentration is μm, nystatin does not inhibit the entry of pedv, which is consistent with park's results. secondly, park et al. added methyl cellulose to block second-cycle infection [ ] , while we added nothing except trypsin in medium. whether these reasons cause two different results needs further study. lipid raft acted as a platform for cell signal transduction and viral invasion, distributed in an island form on the plasma membrane of cells, and was isolated by sucrose density gradient centrifugation. western blotting analysis showed that pedv n protein located in the lipid raft (upper layer) with caveolin- in the cells. as shown in figure b , pedv n protein is also present in the bottom layer when infected with ipec-j cells, which may be due to the different composition of the plasma membranes of the two kinds of cells. we demonstrated that pedv gi subtype gds and gii subtype gds strains could enter vero and ipec-j cells via the clathrin-, caveolae-, and lipid raft-mediated endocytosis pathways. furthermore, we also found that the invasion efficiency of the two strains was different with different endocytosis pathway. these differences between gds and gds strains may be due to the difference of s gene especially the s region of s gene (homology was about %), which is responsible for cell entry and membrane fusion by binding with receptor. the difference of gene may lead to the difference of binding ability or affinity between s protein and receptor, thus leading to the different utilization or initiation efficiency of different endocytosis pathways. however, whether the different gene sequence causes different invasion efficiency between gds and gds strains needs further study. after internalization, viral particles are transported by specific endosomes for membrane fusion. the classical transit route is the endo-/lysosomal pathway, in which endocytic cargoes are transported along endocytic vesicles, early endosomes, and late endosomes-lysosomes. park et al. have confirmed that nh cl and baf-a could inhibit pedv entry [ ] , which is consistent with our results, but needs to be confirmed by different methods. in this study, in addition to chemical inhibitors, we also used sirna interference and cellular localization of virus particles to identify the role of ph and endosomes. the results of this study revealed that pedv entry relied on low ph, which means that internalized pedv particles are transported to endosomes and lysosomes, as demonstrated by the co-localization of viral particles with eea , rab , and lamp . liu et al. [ ] reported that pedv s protein was activated by lysosomal cysteine proteases to activate pedv entry. however, based on the data, we could not conclude that membrane fusion occurred at the lysosomes; more technical methods are necessary to demonstrate the mechanism. in conclusion, studying the internalization and intracellular trafficking mechanism of pedv are important to understand viral pathogenesis and benefit to the development of future therapies strategies. this study demonstrated that both the gi and gii subtypes of pedv enter vero and ipec-j cells via the clathrin-, caveolae-, and lipid raft-mediated endocytosis pathways, but the efficiency of each endocytosis pathway varies depending on the different genotypes and types of cells. the internalized pedv entered the lysosomes through the early and late endosomes. the results of this study provide a theoretical basis for the further understanding of pedv pathogenesis to find new targets of antiviral drugs. virus-like particles 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jurisdictional claims in published maps and institutional affiliations we would like to thank prof. qiang yang, college of veterinary medicine, nanjing agricultural university, china, for providing us with ipec-j cells. we would also like to thank prof. mark mcniven, mayo center for biomedical discovery, usa, for providing us with overexpression vector of wild-type and domain negative mutant of dynamin ii and caveolin- . supplementary information accompanies this paper at https ://doi. org/ . /s - - - . and ipec-j cells were treated with different concentrations of dynasore at °c for h. cck- solution was added to each well at °c for h, and absorptions of nm were detected. dmso was used as a negative control. (b) the vero cells and ipec-j cells were transfected with sidyn, and the second transfection was carried out at h after the first transfection. the inference efficiency was detected by qrt-pcr and western blotting at h after the first transfection. ctrl means control. ** . < p < . ; *** . < p < . ; ****p < . .additional file . clathrin-mediated endocytosis is involved in pedv entry. (a) vero cells and ipec-j cells were treated with different concentrations of cpz at °c for h. cck- solution was added to each well at °c for h, and absorptions of nm were detected. double-distilled water was used as a negative control. (b, c) the vero cells and ipec-j cells were transfected with sichc and sieps , and the second transfection was carried out at h after the first transfection. the inference efficiency was detected by qrt-pcr and western blotting at h after the first transfection. ctrl means control. *p < . ; ** . < p < . ; *** . < p < . ; ****p < . . cells were seeded in -well plates until confluence. cells were pre-treated with μm cpz, mm mβcd, μm nystatin, mm nh cl and nm baf a respectively, at °c for h and incubated with gds (a) and gds (b) strains for h. double-distilled water was used as a negative control. the cells were collected at hpi and detected by immunofluorescence staining against the pedv s protein (green). nuclei were stained with dapi (blue). scale bars indicate μm. cells were seeded in -well plates until confluence. cells were pre-treated with μm cpz, . mm mβcd, μm nystatin, mm nh cl and nm baf a respectively, at °c for h and incubated with gds (a) and gds (b) strains for h. double-distilled water was used as a negative control. the cells were collected at hpi and detected by immunofluorescence staining against the pedv s protein (green). nuclei were stained with dapi (blue). scale bars indicate μm. the authors declare that they have no competing interests.received: october accepted: january