key: cord-314546-fbddxbhd authors: ko, meehyun; jeon, sangeun; ryu, wang‐shick; kim, seungtaek title: comparative analysis of antiviral efficacy of fda‐approved drugs against sars‐cov‐2 in human lung cells date: 2020-08-16 journal: j med virol doi: 10.1002/jmv.26397 sha: doc_id: 314546 cord_uid: fbddxbhd drug repositioning represents an effective way to control the current covid‐19 pandemic. previously, we identified 24 fda‐approved drugs which exhibited substantial antiviral effect against severe acute respiratory syndrome coronavirus 2 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(50) = 0.0022 µm). coronavirus disease 2019 (covid-19) is an emerging infectious disease caused by a coronavirus. 1 the causative virus was named as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) because it is very similar to sars-cov (79.5%) and this virus belongs to the betacoronavirus genus within the coronaviridae family. 2 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-19 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-2. 3 previously, we identified a total of 24 potential antiviral drug candidates from fda-approved drugs using vero cells. 4 since antiviral efficacy could be altered in different cell lines, we developed a new image-based antiviral screening assay with calu-3 cells, a well-known human lung cell line, 5 and compared the antiviral efficacy of the antiviral candidates in between vero and calu-3 cells. calu-3 used in this study is a clonal isolate, which shows higher growth rate compared with the parental calu-3 obtained from the american type culture collection (atcchtb-55). calu-3 was maintained at 37°c with 5% co 2 in eagle's minimum essential medium (emem, atcc), supplemented with 10% heat-inactivated fetal bovine serum (fbs) and 1x antibiotic-antimycotic solution (gibco). sars-cov-2 (βcov/kor/kcdc03/2020) 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-2 were performed at institut pasteur korea in compliance with the guidelines of the knih, using enhanced biosafety level 3 (bsl-3) containment procedures in laboratories approved for use by the kcdc. in our previous drug repositioning study, we identified a total of 24 potential antiviral drug candidates from fda-approved drugs. 4 (table 1) , the ic 50 of remdesivir rather decreased by 10 folds compared with that with vero cells, perhaps due to the low metabolic capacity or prodrug activation rate in vero cells 6 ( table 2 ). 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-19 treatment 7-9 ; however remdesivir seems to be effective for treatment of patients with covid-19 in certain clinical settings. 10 interestingly, the ic 50 values of most drugs in our study increased in varying degrees in calu-3 cells (figures 2a,c) (tables 1 and 3 ). only six drugs showed decreases in ic 50 ( figure 2b ) ( table 2) : 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-3 cells following the discovery that tmprss2, a host protease necessary for priming viral spike glycoprotein, could be a target for covid-19 antiviral development. 11 the discrepancy in ic 50 was specifically remarkable with nafamostat mesylate; the ic 50 decreased by approximately 6000 folds when the drug was used in the sars-cov-2-infected calu-3 cells perhaps due to the dominant role of tmprss2-dependent viral entry in the calu-3 human lung epithelial cells. 12, 13 in addition, the ic 50 of nafamostat mesylate was exceptionally low (0.0022 µm), which indicates that nafamostat mesylate is approximately 600-fold more potent than remdesivir in calu-3 cells. it became more apparent that blood clotting is one of the complicating manifestations in patients with covid-19, 14, 15 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-19 with nafamostat 16 and other in vitro studies 17,18 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-19 patients to nafamostat treatment. in summary, we compared antiviral efficacy of the potential antiviral drug candidates against sars-cov-2 in between vero and calu-3 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-19 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 2019-ncov and naming it sars-cov-2 remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro identification of antiviral drug candidates against sars-cov-2 from fda-approved drugs cultured human airway epithelial cells (calu-3): a model of human respiratory function, structure, and inflammatory responses remdesivir potently inhibits sars-cov-2 in human lung cells and chimeric sars-cov expressing the sars-cov-2 rna polymerase in mice observational study of hydroxychloroquine in hospitalized patients with covid-19 effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 remdesivir for the treatment of covid-19-preliminary report sars-cov-2 cell entry depends on ace2 and tmprss2 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-19 pulmonary embolism in covid-19 patients: awareness of an increased prevalence three cases of treatment with nafamostat in elderly patients with covid-19 pneumonia who need oxygen therapy nafamostat mesylate blocks activation of sars-cov-2: new treatment option for covid-19 the anticoagulant nafamostat potently inhibits sars-cov-2 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-2 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/0000-0003-3954-5908 key: cord-276361-77cylm1o 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: 2004-06-04 journal: biochem biophys res commun doi: 10.1016/j.bbrc.2004.04.083 sha: doc_id: 276361 cord_uid: 77cylm1o 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-1 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, 2002. since then, sars has spread to 32 countries and has resulted in more than 800 deaths from respiratory distress syndrome [1] [2] [3] . an overall estimate of case fatality reached 14-15% as reported by who [4] and the mortality rate in people older than 60 years could be as high as 43-55% [5] . several groups, including the authors, isolated a novel coronavirus from sars patients [2, 6, 7] . 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 [8] . 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 [9] . 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 [10, 11] . 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 [12] . moreover, significant toxicity, such as hemolytic anemia, has been attributed to ribavirin [13] . a few preliminary trials and in vitro data suggested the possibility of treating sars with interferon [14] [15] [16] . other agents including glycyrrhizin and convalescent plasma require further studies [17] . as is well established in the case of hiv-1 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-1 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 e6 cells were maintained in dulbecco's modified eagle's medium supplemented with 10% fbs and glutaminepenicillin-streptomycin solution in 5% co 2 in humidified air at 37°c. the ffm-1 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 24 drugs as follows: nelfinavir, saquinavir, kni-272, tya5, tyb5, ritonavir, lopinavir, indinavir, 4f-benzoyl-tn14003, 4f-benzoyl-te14011, tn14003, t140, tc14012, fc131, t22, sdf-1, vmip-ii, tak-779, sc34, n36, t-20, glycyrrhizin, glycyrrhetic acid, and cardran sulfate. cytopathic effect assay. sars-cov was inoculated into a monolayer of vero e6 cells in 24-well plates at a multiplicity of infection (moi) of 0.01. the plates were incubated at 37°c in 5% co 2 for 3 days and cpe in each well was observed. immunofluorescence assay. the vero e6 cells in 24-well plates were infected with sars-cov at the moi of 0.01. the infected cells were fixed with methanol 24 h after infection and incubated at room temperature for 1 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 30 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-1 rna, we performed real-time rt-pcr with the primers and the probe as follows: orf1-f, agctacgagcaccagacacc; orf1-r, actttgggcattccccttt; orf1-probe, tcgaaa ttaagagtgccaagaaatttgacacttt. the fluorescence intensity generated from the probe was detected by the abi-7700 sequence detector system (applied biosystems). mtt assay. vero e6 cells in 96-well plates were infected with sars-cov at the moi of 0.01. after 36 h of culture, cells were incubated for 4 h in the presence of 0.5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (mtt). formazan crystals were dissolved with 100 ll of 0.04 n hcl-isopropyl alcohol (acid isopropanol) and absorbance at 570 nm was measured with a reference wavelength of 655 nm. time-of-addition experiments. drugs, including nelfinavir, were added to cultures of vero e6 cells at the time of infection or 3 h after infection. samples were processed for a quantitative rt-pcr assay and an immunofluorescence assay 24 h after infection. the cytopathic effect of infected cells was analyzed 36 h after infection. entry inhibition assay. vero e6 cells were pretreated with each drug for 3 h, and sars-cov was inoculated at the moi of 0.1. cells and viruses were incubated for 3 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, 18s ribosomal rna was quantified with the primers and the probe as follows: 18s-f, gtaacccgttgaaccccatt; 18s-r, ccatccaatcggtagtagcg; and 18s-probe, tgcgtt gattaagtccctgccctttgta. we screened our chemical library and found that nelfinavir could inhibit sars-cov replication in vero e6 cells. nelfinavir clearly inhibited the cytopathic effect (cpe) induced by infection with sars-cov (fig. 1a) . 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. 1b) . 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. 2) . by the use of mtt assay, we determined the concentration of the compound that reduced cell viability to 50% (cc 50 ), the concentration of the compound required for inhibition of cpe to 50% of the control value (ec 50 ), and the selectivity index (si). nelfinavir inhibited sars-cov replication at non-toxic doses with an approximate si of 300, while the other inhibitors against hiv-1 protease (ritonavir, lopinavir, saquinavir, indinavir, tya5, tyb5, and kni-272) did not affect the replication of sars-cov (table 1 ). 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. 1a and b and 2) . when this drug was added at the time of infection or 3 h after infection, it was still able to block the cpe induced by sars-cov infection (fig. 3a) . addition of nelfinavir at various timings inhibited the expression of viral antigens in vero cells as shown by ifa (fig. 3b) . nelfinavir blocked the production of virions when used to treat the cells at the time of infection or 3 h after infection (fig. 4) . the other protease inhibitors including ritonavir had no effect on replication of sars-cov cc 50 , cytotoxic concentration of the compound that reduced cell viability to 50%. mean (standard error) of three assays was calculated for each drug. ( figs. 3a and b and 4) . 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. 5) , 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 [18] [19] [20] , 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-1 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) [21] . when used in this manner, 50-75% 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 200 mm à3 cd4(+) lymphocytes at 12 months of therapy [22] [23] [24] [25] . the most common side effect of nelfinavir is mild diarrhea, which is observed in 15-20% of patients [26] . 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 e6 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 144 patients with sars in the greater toronto area treatment of sars with human interferons interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study pegylated interferon-alpha protects type 1 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-1-infected subjects according to cd4 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, ag1343): a potent, orally bioavailable inhibitor of hiv-1 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-102246-2lmq9s4l authors: salvadori, marcia r.; yamada, aureo t.; yano, tomomasa title: morphological and intracellular alterations induced by cytotoxin vt2y produced by escherichia coli isolated from chickens with swollen head syndrome date: 2001-04-01 journal: fems microbiology letters doi: 10.1016/s0378-1097(01)00090-8 sha: doc_id: 102246 cord_uid: 2lmq9s4l abstract recently, a novel verocytotoxin named vt2y was described which belongs to the stx family and is produced by escherichia coli isolated from domestic poultry with swollen head syndrome (shs). the vt2y 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 15 min and was maximal at 1 h after treatment with vt2y. 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 [16] 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 [19] . 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 [16, 23] . since the syndrome was described in south africa, in 1984, 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 [6, 18, 20, 26] . vtec strains produce one or more vts: vt1, and several variants of vt2. the vt2v is produced by e. coli isolated from pigs with ed [11] . recently par-reira and yano [20] described a novel cytotoxin called vt2y and sequencing of a 1560-bp dna fragment of vt2y showed that it contained the stx gene of shigella dysenteriae, but the region 300 bp upstream of the start of the gene for the stxa subunit was identical to the corresponding sequence for e. coli o111:h-, except for 1 nucleotide, suggesting that vt2y is a member of the stx family [22] . the aim of this study is to illustrate the morphological and intracellular alterations induced in vitro by cytotoxin vt2y in vero, hela, cho, hep-2, pck and cef cell lines. 2.1. bacterial strains e. coli shs-8 strain isolated from chickens with shs was used as a source of vt2y [21] . e. coli strain k12c600 was used as a negative control and strain j2 (o157 :h-vt2) was used as a source of vt2 (kindly donated by yoshifumi takeda, japan) in these experiments. all e. coli strains were stored at 320³c in luria^bertani (lb) [14] broth to which 15% glycerol was added. the cytotoxin vt2y was puri¢ed [21] , and protein content was assayed as described by bradford [2] . the cytopathic e¡ect (cpe) of puri¢ed vt2y was examined in vero (african green monkey kidney), hela (human epitheloid cervical carcinoma), cho (chinese hamster ovary), hep-2 (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 [10] . the cells were grown at 37³c in eagle's minimal essential medium (emem) with earle's salts (cultilab, campinas, sp, brazil), supplemented with 10% fetal bovine serum (fbs, sigma, st. louis, mo, usa), penicillin (1000 u ml 31 ) and streptomycin (250 wg ml 31 ). to determine cytotoxic activity [18] , 100 wl of medium and 2u10 5 cells per well were placed in 96-well plastic plates (costar, cambridge, ma, usa). after 24 h, culture medium was aspirated and 150 wl of fresh emem without fbs, and 50 wl (9 ng ml 31 ) of puri¢ed vt2y were added to each well. the plates were incubated at 37³c in a 5% co 2 atmosphere for 24 h. morphological changes were observed over the 24-h period and the test was scored as positive if more than 50% of the cells exhibited cytotoxic e¡ects. control e. coli strains were j2 (o157:h-vt2) and k12c600. the cellular viability was determined using a neutral red (2-amino-3-methyl-7-dimethyl-amino-phenazoniumchloride) assay [1] . the neutral red assay was performed in vero, hela, cef, pck and cho cells. puri¢ed vt2y cytotoxin was applied to the cells and after 1 h, the medium was substituted with 0.2 ml of neutral red solution (50 wg ml 31 ) per well and the plate incubated at 37³c in 5% co 2 for 3 h, to allow for dye incorporation. afterwards, the dye medium was removed and each well was rapidly washed with formol^calcium solution (10% formaldehyde and 10% calcium chloride) to remove unincorporated neutral red; 0.2 ml of solution containing 1 ml acetic acid with ethanol 50% was added to each well and the plate kept for 15 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 100% with viable cells. the color reaction was measured with an elisa plate reader (multiskan bichromatic) at 540 nm. the cd 50 , was de¢ned as the minimum amount of vt2y required to kill 50% of the cells (1 cd 50 ), and was determined from the toxin concentration resulting in 50% neutral red absorbance compared to control cells as 100%. leakage of the cytosolic enzyme ldh, was assessed [15] to investigate whether vt2y a¡ected the integrity of the plasma membrane of vero cells. medium from vero cells treated with 3 cd 50 of vt2y or without vt2y was collected after 15, 30, 60, 90 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 1 ml freshly prepared potassium phosphate bu¡er (0.1 m, ph 7.4) and the mixture was equilibrated for 30 min at 37³c. the enzyme activity was determined spectrophotometrically and the rate of enzyme leakage min 31 was calculated and expressed as percentage of total ldh activity as compared to untreated control cells. vt2y in vero, hela, cef, pck and cho cells the vero, hela, cef, pck and cho cells were grown on coverslips in 24-well plates using 10 4 cells ml 31 per well in a volume of 1 ml. the plates were incubated at 37³c in 5% co 2 . after 24 h, culture medium was aspirated and replaced with 1 ml of fresh medium. then, 3 cd 50 vt2y 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 10% formaldehyde solution in pbs for 1 h, then washed with distilled water and stained with 0.025% toluidine blue in mcilvaine ph 4.0 bu¡er [13] . the coverslips were washed for 15 min with distilled water, air-dried, cleared in xylene and mounted using entellan (merck, germany). dna fragmentation in vt2y-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 vt2y was cytotoxic to vero, hela, cef, pck and cho cells, but not for hep-2 cells. by light microscopy the cytotoxin-a¡ected cells appeared round and shrivelled, and the cells were dead within 1 h. the neutral red assay showed that vero cells were more sensitive than other cells tested since 50% cell death was observed in the presence of 0.1 ng ml 31 vt2y. a linear increase in the percentage of dead cells was found as protein concentration was increased within the range indicated in fig. 1. after 15 min minimal (5%) ldh leakage was observed as compared to the control (fig. 2) , but by 60 min an increase to 46% above control ldh leakage had occurred. the cytotoxin vt2y 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 15 min. the e¡ects became more pronounced with time to a¡ect the majority of cells. by 1 h, 100% cell death was observed (data not shown). when cells were cultured in the presence of vt2y, they showed condensed chromatin (fig. 3b) , cleaved nuclei (fig. 3c ) and cytoplasmic blebbing (fig. 3d) , ¢ndings typical of apoptosis. we also observed the presence of cytoplasmic vacuoles in conjunction with apoptotic fea-tures (fig. 3c) . however, none of these features occurred in control cells (fig. 3a) . 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. 4b) . the same vero cells clearly incorporated biotinylated nucleotides, indicating the occurrence of dna fragmentation induced by vt2y, in contrast with control cells (fig. 4a ). programmed cell death, or apoptosis, is de¢ned by the cell's ultrastructural morphology [7] 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 [12] . recently, reports have claimed that vts induce programmed cell death, apoptosis, in cell lines such as vero, mdck [5, 25] and others. in our studies, we showed evidence of morphological alterations characteristic of apoptosis (fig. 3 ) in three mammalian cell (vero, hela and cho) and two avian cell lines (pck and cef), but not in hep-2 cells. the most sensitive line to vt2y was vero cells (fig. 1) , probably due to receptor speci¢city of the cells, as it is known that variants of vt2 are more cytotoxic to vero cells than hela cells [4] ; the receptor for vt1 and vt2 is globotriosyl ceramide (gb3) [9] , whereas the preferred receptor for vt2v is globotetraosyl ceramide (gb4) [24] . it will be interesting to study the receptor for this toxin on the avian cells pck and cef, since vt2y is produced by avian e. coli strains. we also observed the presence of multiple vacuoles in the cytoplasm of these cell lines (fig. 3c) , that simultaneously showed apoptotic features induced by vt2y cytotoxin. the microscopic nuclear fragmentation and incorporation of nucleotides shown by the tunel method occurred following vt-treatment of vero cells (fig. 4) . by performing the ldh assay we veri¢ed a similar time-response using 3 cd 50 vt2y; marked apoptosis e¡ects were also observed when cells were assessed using a toluidine blue assay. based on these data, we suggest that the vt2y-induced cell death occurs by the apoptotic pathway after plasma membrane damage. interestingly morphological evidence of apoptosis was observed within 15 min and was maximal within 1 h of toxin administration to vero cells, which is considerably more rapid than previously described for apoptosis induced by vt2 in epithelial cells [8, 27] . ed in pigs is caused by the toxin variant vt2v produced by e. coli [11] . this disease is characterized by anorexia, edema of the eye-lids and neurological abnormalities, such as lack of coordination and paralysis [3] ; these characteristics, are similar in some respects to those shown by chickens with shs [17] . here we observed that vt2v induced apoptosis in vero cells, but in contrast to vt2y the morphological alterations of apoptosis were detected 3 h after vt-treatment (data not shown). from this evidence, a histopathological study of the action of vt2y 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-90) 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-1 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 1 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. 4th 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 3-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-004825-cdvnqfjz authors: castilla, v.; mersich, s. e.; candurra, n. a.; damonte, e. b. title: the entry of junin virus into vero cells date: 1994 journal: arch virol doi: 10.1007/bf01321064 sha: doc_id: 4825 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 5.5 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 60-64 kda) and an external glycoprotein (gp38, mw 38 kda) [20] . other minor proteins have also been described in purified virions or in infected cells [5, 6, 10] . 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 [27] . in humans, jv induces argentine haemorrhagic fever, an endemoepidemic disease with hematologic and neurologic signs, which mainly affects male rural workers [-27] . 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 [-2, 3] . 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 [8] . 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 [15] . 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 [1, 9, 14] . 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 [15] . 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 5% calf serum and gentamycin. the iv4454 strain of jv [4] was used. stock solutions of amantadine (specia), ammonium chloride (fisher company), chlorpheniramine (schering-plough) and procaine (schering-plough) at concentrations of 5, 500, 100 and 50mm, respectively, were prepared in culture medium and sterilized by filtration. ammonium chloride (15mm) was added to vero cells 1 h before infection with jv or at 0, t, 3, 5 or 8 h post-infection (p.i.). in all cases supernatant cultures were harvested at 24 h p.i. and extracellular virus yields were determined by plaque assay. vero cells were infected with jv (virus inoculum: 2 × 105 pfu) in the presence or in the absence of 15mm ammonium chloride. after adsorption for 0, 10,20,30, and 60rain at 4 °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 1. after 30 rain adsorption at 4 °c two washes were done to remove excess inoculum and then cultures were warmed to 37 °c for various times in the presence or absence of 15 mm ammonium chloride. cultures were subsequently treated with proteinase k (0.25 mg/ml) in pbs for 30 min at 4 °c in order to remove external virus. protease treatment was stopped by the addition of 2mm phenyl methyl sulfonyl fluoride (pmsf), 3~ bovine serum albumin (bsa) in pbs. cells were centrifuged 10 min at 2 000 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 1) and 15 mm ammonium chloride was added to the culture medium after adsorption. at 24 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 [23] . veto cell monolayers in 24-wetl plates were adsorbed for 1 h at 4 °c with jv in mem containing 0.2~/o bsa, 15 mm hepes, ph 7.5. in a set of cultures cells were washed twice with pbs and then warmed to 37 °c by the addition of pre-warmed medium buffered with 10 mm hepes and 10 mm 1-4-piperazinediethene-sulfonic acid (pipes) at ph 5.5, 5.8, 6.1, 6.3, 6.6 and 7.5, in the presence or in the absence of 15 mm ammonium chloride. after 3 h of incubation at 37 °c, cells were washed twice with pbs and mem containing 0.7~ methylcellulose, ph 7.5, was added. in other set of cultures, after adsorption cells were washed with pbs and incubated at 37 °c for 2 rain in mem buffered at different ph as indicated above. then, cells were washed with pbs and mem at ph 7.5 containing or not 15ram ammonium chloride was added. after 3h of incubation cultures were overlaid with methylcetlulose as above. in both set of cultures plaques were counted after 7 days of incubation and percent of inhibition was calculated. vero cells were infected with jv at a moi of 1. at 48 h after infection, medium was removed and cells were incubated in mem buffered at ph 5.5 or 7.5 as indicated above. then, monolayers were stained with giemsa and syncytia containing more than 10 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 15 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. 2) . 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 5 and 8 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 1 , ammonium chloride had no effect on jv adsorption since the titers of virus adsorbed at 4 °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 5%. to investigate whether ammonium chloride affects jv internalization, virus adsorbed cells at 4 °c were warmed to 37 ~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 30 min of incubation at 37 °c and the uptake was apparently complete after 1.5 h (fig. 3) . 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 30min 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 15mm ammonium chloride was 98.8% (fig. 4) . 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 (5.5-7.5) (data not shown). a high degree of inhibition was observed when cultures were incubated in medium containing ammonium chloride at ph 7.5, 6.6 and 6.3 during 3 h, while there was only 31.5 and 22.3 % inhibition at ph 6.t and 5.8, respectively (fig. 5) . 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 2 min and then incubated for 3 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 [26] . under these experimental conditions, ammonium chloride inhibition was partially reversed at ph 5.5 (fig. 5 ). 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 5.5 or 7.5 was quantitated. cell fusion was observed at 48 h p.i. but only at ph 5.5, there being more than 10 nuclei in the syncytia whereas no polykaryon formation was seen at neutral ph (fig. 6 ). 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 [18] . we have demonstrated here (figs. 1, 4) . 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. 2) . however, virus attachment which occurred at 4 °c was not inhibited as it is shown in table 1 . virions b o u n d to cells at 4°c are compelled to internalize when the temperature of incubation is raised to 37 °c. when jv internalization, the next stage of the viral cycle, was studied a significant inhibition was observed during the first 372 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. 3 .) 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 6.1 (fig. 5) , 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 [15] . for some viruses such as semliki forest virus, fusion occurs at a ph 6.2 in early endosomes, whereas influenza virus x-31 requires ph 5.3 and fuses in the late endosomes [12, 24, 25] . for jv entry, fusion seems to occur in endosomes where the ph is 6.1 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 [7], rubella virus [17] , west nile virus [13] and tick-borne encephalitis virus [11] . it is possible to induce fusion in some model systems by mimicking the low ph intracellular conditions. in particular, mann et al. [16-1 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 5.5 producing polykaryocytes in which over 40% of the cells in the monolayer participate (fig. 6) . thus, this result offers indirect support for the conclusion that the route of jv entry is ph-dependent in vero cells. major expression of gp38, the main external envelope glycoprotein [19] , was observed in the surface of jv infected cells at 48 h p.i., by immunofluorescence assay (data not shown). thus, gp38 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 c167, a host-range mutant of jv, with an alteration in gp38 detected by peptide mapping and a blockade in adsorption-penetration pathway [21, 22] . 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 gp38 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 (t990) a comparison of junin virus strains growth characteristics, cytopathogenicity and viral polypeptides polypeptide synthesis in junin virus infected bhk-21 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 (t990) 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 (t989) 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 t6cnicas (conicet) and universidad de buenos aires. e.b. damonte is member of the research career from conicet. key: cord-279316-xz7aawem authors: mizutani, t. title: signal transduction in sars‐cov‐infected cells date: 2007-04-23 journal: ann n y acad sci doi: 10.1196/annals.1408.006 sha: doc_id: 279316 cord_uid: xz7aawem 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 p38 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). 26, 34 in late 2002, sars spread from china to more than 30 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. 15, 23, 40 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. 1) . 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 e6, 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 e6 cells induces apoptosis, detected by dna fragmentation and caspase activation. 28, 42 p38 mapk is strongly activated by stress and inflammatory cytokines. mouse hepatitis virus (mhv), a prototype coronavirus, was able to induce activation of p38 mapk into virus-infected cells. 1 p38 mapk activation in cd14 monocytes was also observed in sars patients. 24 phosphorylation of p38 mapk was significantly upregulated at 18 h postinfection (h.p.i.) in sars-cov-infected vero e6 cells. 28 cytopathic effects of sars-cov-infection were partially prevented by adding the p38 mapkspecific inhibitor, sb203580, to the cells. although p38 mapk can promote both cell death and survival, 10 the results of inhibitor studies indicated that the p38 mapk signaling pathway may be involved mainly in cell death in sars-cov-infected vero e6 cells. several downstream targets of p38 mapk were phosphorylated in virus-infected cells, and sb203580 effectively inhibited phosphorylation of these proteins in sars-cov-infected cells. mapkactivated protein kinase (mapkapk)-2, which is activated in response to stress and growth factors, 12, 13 was phosphorylated in virus-infected cells. hsp-27, which is a substrate of mapkapk-2 and is known to show antiapoptotic activity by inhibiting apoptosome formation, 14 survival factors, 20 camp response element-binding protein (creb), and activation of transcription factor (atf)-1, was also phosphorylated in virus-infected cells. phosphorylation of hsp-27, creb, and atf-1 may induce an antiapoptotic environment in sars-cov-infected cells. nucleocapsid (n) protein was able to induce phosphorylation of hsp-27 and creb in transfected cells as described below. 36 the translation initiation factor, eukaryotic initiation factor 4e (eif4e), is known to enhance translation rates of cap-containing mrnas. 16 the phosphorylation of eif4e was utilized to promote virus-specific protein synthesis in the case of mhv. 1 although the levels of phosphorylated eif4e were increased by sars-cov infection, the activated eif4e was not advantageous for viral protein synthesis as demonstrated by the similar kinetics of viral protein accumulation in infected vero e6 cells in the presence and absence of sb 203580. there may be other substrates of p38 mapk that are inducible on apoptosis of vero e6 cells caused by sars-cov infection. as each downstream target molecule of p38 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 p38 mapk is important for the development of anti-sars-cov drugs. extracellular signal-regulated kinase (erk) 1/2 was phosphorylated in sars-cov-infected vero e6 cells, 27 whereas erk1/2 was downregulated in n protein-expressing cos-1 cells as described below. 36 jnk c-jun n-terminal kinase (jnk) was phosphorylated in sars-cov-infected vero e6 cells. 27 a recent study indicated that persistent infection was established after most of the sars-cov-infected vero e6 cells had died by apoptosis. 30 sp600125, an inhibitor of jnk, and ly294002, an inhibitor of phosphatidylinositol 3-kinase (pi3k), inhibited the establishment of persistence, whereas pd98059, an inhibitor of mek1/2, and sb203580, an inhibitor of p38 mapk, did not. thus, two signaling pathways of jnk and pi3k are important for the establishment of persistence in vero e6 cells. in vero e6 cells, signal transducer and activator of transcription (stat) 3 is constitutively phosphorylated at tyrosine (tyr)-705 and is slightly phosphorylated at serine (ser)-727. 27 however, sars-cov replication induced dephosphorylation of stat3 at tyr-705. as stat3 is a major transcription factor activated in response to cytokines, such as interleukin-6 (il-6) and il-10, inhibition of stat3 signaling by dominant negative and antisense oligonucleotides against stat3 resulted in decreases in cell viability and apoptosis. 17, 31 thus, stat3 is thought to act as an antiapoptotic transcription factor. although inhibitors of mek and jnk had no effect on the phosphorylation status of stat3 in virus-infected cells, two inhibitors of p38 mapk (sb203580 and sb202190) partially inhibited dephosphorylation of stat3 at tyr-705, suggesting that the p38 mapk signaling pathway is upstream of tyr-705 dephosphorylation of stat3 in vero e6 cells. the level of stat3 phosphorylation at ser-727 was increased in virus-infected cells. although the effect of ser-727 phosphorylation on the function of stat3 remains unresolved, it was reported that phosphorylation of ser-727 of stat3 negatively modulated its tyr phosphorylation. 6 interestingly, tyr-705 dephosphorylation and ser-727 phosphorylation showed almost the same timing in sars-cov-infected cells. stat3 phosphorylated at tyr-705 was localized mainly in the nucleus in mockinfected cells, whereas stat3 disappeared from the nucleus in virus-infected cells. as stat3 acts as an activator of transcription in the nucleus, stat3 may lack its transcriptional activity in sars-cov-infected vero e6 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 stat1 and stat2 via jak1 and tyk2, whereas the ifn-gamma receptor recognizes stat1 via jak1 and jak2. signal-transducing adaptor molecule 1 (stam1) was upregulated in sars-cov-infected vero e6 cells. 25 as stam1 is known to associate with jak2 and 3 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 pi3k signaling pathway (including akt) plays important roles in establishing persistent infection by sars-cov. akt is phosphorylated at both ser-473 and threonine (thr)-308 residues via a pi3k-dependent mechanism on stimulation by growth factors, insulin, and hormones. 39 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 3ß (gsk-3ß), caspase-9, and bad. 2, 8, 9 in sars-cov-infected confluent vero e6 cells, ser-473 of akt was phosphorylated at 8 h.p.i. and maximal phosphorylation was observed at 18 h.p.i., whereas phosphorylation of thr-308 was not observed. as akt is thought to show its full activity when phosphorylated at both ser-473 and thr-308, the total activity of akt may be low in vero e6 cells. therefore, the phosphorylated akt in sars-cov-infected cells cannot prevent apoptosis. interestingly, tissue inhibitor of metalloproteinase 2 (timp2), which activates ras and leads to ras/pi3k complex formation, was shown to be downregulated in virus-infected vero e6 cells at 12 h.p.i. by microarray analysis. 25 the acute ser dephosphorylation of akt at 24 h.p.i. after tentative phosphorylation in virus-infected cells may be due to downregulation of timp2. 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, 32 is thought to be one of the most important pkc, because pi3k-dependent activation of pkc mediates bcell survival by nerve growth factor. 22 akt has been reported to interact with pkc . 21 pkc was phosphorylated in sars-cov-infected vero e6 cells, 27 suggesting that this molecule is activated as an antiapoptotic response to sars-cov infection. human intestinal epithelial caco-2 cells, which are highly permissive to sars-cov, have been used for analysis of cellular gene expression by microarray analysis. the proinflammatory chemokines, interleukin 8 (cxcl8), and interferon-␥ -inducible protein 10 (cxcl10), were upregulated in sars-cov-infected caco-2 cells. 7 these two chemokines are regulated by ap-1 and nf-b, which were also activated by viral infection. cxcl10 levels were significantly elevated in the blood of sars patients 38, 41 and in macrophages in vitro. 5 as described above and below, creb was phosphorylated in virus-infected vero e6 cells and n protein-expressing cos-1 cells. the genome of sars-cov is approximately 30 kb in length and contains 14 potential open-reading frames (orfs), including nine viral proteins with no homologues in other coronaviruses. the n protein, which is a 423 amino acid predicted phospho-protein of 46 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 209 amino acid region 35 and was able to induce apoptosis of cos-1 cells in the absence of growth factors. 36 both jnk and p38 mapk were upregulated in the transfected cells, whereas erk and akt were downregulated. activated p38 mapk by n protein induced actin reorganization into cells in the absence of growth factors. the downstream targets of p38 mapk, mapkapk-2, and hsp-27, were phosphorylated in the cells as well as in the virus-infected vero e6 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-3 and -7 were activated only in the absence of growth factors in n-expressing cells. however, sb203580, an inhibitor of p38 mapk, failed to inhibit caspase-3 activation. thus, there are two independent functional properties of n protein, p38 mapk-dependent actin reorganization, and caspase activation. another group showed that the levels of transcription factors, c-fos, atf2, creb, and fosb, are increased by expression of n in vero and huh-7 cells. 18 spike (s) protein of sars-cov also induced activation of ap-1 and il-8 promoter, possibly via activation of mapks, in a549 and hfl-1 cells. 4 as high serum levels of il-8 were observed in patients in the acute stage of sars, 19 activation of the il-8 promoter via ap-1 induced by s protein may explain these clinical observations. the binding of s protein to a viral receptor, angiotensin-converting enzyme (ace)-2, may be a trigger for activation of these mapks. two unique proteins of sars-cov, 3a (u274, x1) and 7a (u122, x4), have been studied. the 3a protein is located mainly in the golgi apparatus and contains three transmembrane regions, 43 whereas 7a encodes a protein of 122 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, 7a is localized to the perinuclear region in both sars-cov-infected and -transfected cells. 11 the overexpression of 7a, but not of 3a, induced apoptosis via a caspase-dependent pathway. 37 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 p38 mapk is involved in the regulation of il-8 in vivo. both akt and p38 mapk are keys for determination of cell survival or death in sars-cov-infected cells in vitro. activation of the p38 mapk signaling pathway and dephosphorylation of stat3 via p38 mapk induced by sars-cov infection have partially proapoptotic roles in vero e6 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 p38 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 p38 mitogen-activated protein kinase activation promotes interleukin-6 production and virus replication in cultured cells regulation of cell death protease caspase-9 by phosphorylation mammalian map kinase signalling cascades induction of il-8 release in lung cells via activator protein-1 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 stat3 serine phosphorylation by erk-dependent and -independent pathways negatively modulates its tyrosine phosphorylation high-dose hydrocortisone reduces expression of the pro-inflammatory chemokines cxcl8 and cxcl10 in sars coronavirus-infected intestinal cells inhibition of glycogen synthase kinase-3 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 (u122) of the severe acute respiratory syndrome coronavirus hemopoietic growth factors with the exception of interleukin-4 activate the p38 mitogen-activated protein kinase pathway interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of hsp27 hsp27 and hsp70: potentially oncogenic apoptosis inhibitors organization and regulation of mitogenactivated protein kinase signaling pathways eif4 initiation factors: effectors of mrna recruitment to ribosomes and regulators of translation constitutive activation of stat3 signaling abrogates apoptosis in squamous cell carcinogenesis in vivo activation of ap-1 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 p38 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 stat3 in sars coronavirus-infected vero e6 cells phosphorylation of p38 mapk and its downstream targets in sars coronavirus-infected cells importance of akt signaling pathway for apoptosis in sars-cov-infected vero e6 cells jnk and pi3k/akt signaling pathways are required for establishing persistent sars-cov-infection in vero e6 cells constitutive activation of stat3 in human prostate tumors and cell lines: direct inhibition of stat3 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 209 amino acid interaction domain the sars coronavirus nucleocapsid (n) protein induces actin reorganization and apoptosis in cos-1 cells overexpression of 7a, 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-1 plus corticosteroids protein kinase b and rac are activated in parallel within a phosphatidylinositide 3oh-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 e6 cells subcellular localization and membrane association of sars-cov 3a 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-267446-rpv19oy6 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: 2011-06-12 journal: arch virol doi: 10.1007/s00705-011-1044-6 sha: doc_id: 267446 cord_uid: rpv19oy6 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 50% mortality among suckling piglets and reduced weight among fattening pigs [10] . although the structural and pathological properties of pedv are similar to those of other group 1 coronavirus, including human coronavirus 229e (hcov-229e), transmissible gastroenteritis virus (tgev) and feline infectious peritonitis virus, many biological issues, such as the role of trypsin in infection, remain unresolved [7, 12, 36] . the first successful propagation of pedv in cell culture was done by supplementing the vero cell culture medium with trypsin [15] . 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 [20, 21] . on the other hand, another study reported the successful propagation of the p-5 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 [17] . 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 2 and 3 coronavirus. for example, infection by severe acute respiratory syndrome coronavirus (sars-cov) and murine hepatitis virus strain 2 (mhv-2) requires proteolytic cleavage in their target cells, which is mediated by trypsin-like proteases [24, 29, 32] . the situation for group 1 coronavirus is unclear. recently, the s protein of the group 1 coronavirus hcov-229e 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 [18] . 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 2 coronaviruses, sars-cov and mhv. african green monkey kidney cells (vero, ccl-81) were prepared in minimum essential medium (mem, gibco) supplemented with 5% fetal bovine serum (fbs, gibco). the cell-adapted strain of the korean pedv isolate, kpedv-9, was propagated as described elsewhere [15] , with some modifications. briefly, vero cells were inoculated with kpedv-9 at a multiplicity of infection c1 and cultured in serum-free mem at 37°c, 5% co 2 for 48-60 h. the supernatant was harvested and then clarified by centrifugation at 12,000 g for 10 min at 4°c. concentration and partial purification were performed by ultracentrifugation under a 20% sucrose cushion at 26,000 rpm for 3.5 h. the pellet was resuspended in 10 mm phosphate-buffered saline (pbs, ph 7.4) and stored at -70°c. mouse polyclonal antibodies against pedv were generated by immunizing 6-week-old female balb/c mice (samtako) intraperitoneally with 1 9 10 5 focus-forming units (ffu) of purified kpedv-9 emulsified in an equal volume of complete freund's adjuvant (sigma-aldrich) on day 1 and incomplete freund's adjuvant (sigma-aldrich) on days 14, 21 and 28. whole blood was collected from the retro-orbital sinus on days 0 and 35 and centrifuged at 1500 g for 10 min to separate the sera. cultured vero cells were inoculated with kpedv-9 as described above and allowed to adsorb for 2 h at 37°c. the vero cells were washed twice with pbs and cultured in serum-and trypsin-free mem or mem containing trypsin (10 lg/ml, sigma-aldrich). at 8, 12, 24, and 48 h postinoculation (hpi), culture supernatants were collected for titration in a focus-formation assay, and cells were fixed with 4% formaldehyde in pbs for 30 min and permeabilized with 1% np-40 (sigma-aldrich) in pbs, followed by immunocytochemistry using mouse anti-pedv polyclonal sera [8] . 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. 1 . to investigate the effects of proteolytic cleavage of the surface protein of vero cells and free virions by trypsin, vero cells or kpedv-9 were pre-treated with trypsin prior to infection. trypsin treatment was performed for 30 min at 37°c prior to inoculation, and enzyme activity was neutralized with 2 lg/ml aprotinin (sigma-aldrich). trypsin-pretreated vero cells were inoculated with kpedv-9, and untreated vero cells were inoculated with trypsin-pretreated kpedv-9. after a 2-h incubation to allow adsorption, cells were washed three times with pbs and then cultured in serum-free mem without trypsin for 24 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-9. vero cells were inoculated with kpedv-9 in the presence of various concentrations of trypsin (5, 10, 20, 40 and 80 lg/ml) during the adsorption period and were cultured in serum-free mem at 37°c for 24 h. to investigate the effect of trypsin on the budding stage of pedv infection, kpedv-9-infected vero cells were prepared by inoculating them with purified kpedv-9 and then cultured in mem for 20 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 10 min. after neutralization of trypsin by the addition of aprotinin, cells were cultured for an additional 4 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 3,3'-diaminobenzidine tetrahydrochloride dihydrate (dab, vector labs). all specimens were observed under an inverted microscope. ultrapurified kpedv-9 was treated with various concentrations of trypsin at room temperature (rt) for 10 min and then analyzed by western blotting. vero cells were infected with kpedv-9 in the absence of trypsin for 24 h, and kpedv9-infected vero cells then were harvested and treated with trypsin for 10 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 8% 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-1000plus (fujifilm). statistical analysis was performed using spss, version 7.5, 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 [15] . 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-9 in vero cells in the presence or absence of trypsin supplementation was compared. as shown in fig. 2 , detectable levels of progeny virions were observed from 8 hpi in both trypsin-and nontrypsin-supplemented media. at 12 hpi, the titer was higher (3.09 9 10 3 ffu/ml) in trypsin-supplemented samples than in non-trypsin-supplemented samples (1.08 9 10 3 ffu/ml). the rate of virus production in trypsin-supplemented cultures was also significantly higher than in trypsin-free cultures (virus titer of 1.83 9 10 5 and 1.65 9 10 4 ffu/ml at 24 hpi, respectively). even at 48 hpi, the titer in the trypsinfree cultures only reached peak titer levels of 4.52 9 10 4 ffu/ml, which was significantly lower than the peak titer attained at 24 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 1 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-9 prior to inoculation did not significantly differ from non-trypsin-treated virus after 20 hpi (1.24 9 10 4 and 1.32 9 10 4 ffu/ml, respectively) (fig. 3) . 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 10 lg/ml trypsin for 30 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-9 in serum-and trypsin-free media for 2 h and then washed twice to remove un-bound kpedv-9. the cell-bound kpedv-9 was treated with different concentrations of trypsin for 10 min, and the titers of penetrating and produced virus were determined. when the concentration of trypsin in the medium was increased from 5 lg/ml to 80 lg/ml, the number of pedv penetrating the vero cells also increased from 4 9 10 2 ffu/ml to 9 9 10 2 ffu/ml. the enhanced trypsin-mediated penetration during initial infection resulted in an increase in virus titer at 24 hpi, from 3.0 9 10 4 ffu/ml to 1.0 9 10 5 ffu/ml (fig. 4) . 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-9-infected vero cells were prepared by inoculation for 20 h. the cells were washed extensively and treated with various concentrations of trypsin for 10 min prior to continuing cell cultivation in fresh serum-and trypsin-free medium. kpedv-9-infected vero cells did not show visible signs of syncytium formation in the absence of trypsin, while kpedv-9-infected vero cells treated with 5, 10 and 20 lg/ml trypsin at 20 hpi contained multiple syncytia (fig. 5a) . without trypsin treatment, the virus titer at 4 hpi was 1.4 9 10 3 ffu/ml while kpedv-9-infected vero cells treated with 5, 10, 20, 40 and 80 lg/ml trypsin showed virus titers of 2.1 9 10 3 , 3.0 9 10 3 , 3.5 9 10 3 , 4.1 9 10 3 and 6.7 9 10 3 ffu/ml, respectively (fig. 5b) . 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. 3 and 4 . 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-9-infected vero cells that had not been treated with trypsin was apparent as a species of about 220 kda, which represented the glycosylated native s protein (fig. 6) . in ultrapurified virus, only this protein species was detected, even after trypsin treatment, while 140-kda and 125-kda proteins, likely trypsin-cleaved s protein, were detected in trypsin-treated kpedv-9-infected vero cells (fig. 6) . 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 [23, 24, 32] . however, this fig. 4 enhancement of cellattached kpedv-9 penetration by trypsin. vero cells were inoculated with kpedv-9 for 2 h and then washed to remove unbound kpedv-9. only cellattached kpedv-9 was treated with trypsin for 30 min, and penetrated virus (j) and progeny virus were titrated after 24 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 24 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 [6, 26] . the activated fusion protein undergoes conformational changes that induce fusion of the viral envelope and host membranes [3, 27, 30] . this process usually occurs during the period between virus maturation and virus attachment to the host receptors [25, 33] . after the fusion process, the viral core, including the viral genome, is transported into the cytoplasm where uncoating and replication ensue [22] . 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 [19] . in cell culture, the protease cleavage that is required for virus propagation is carried out by exogenous proteases such as trypsin or pancreatin [1, 15, 28] . these exogenous proteases induce syncytium formation by activating the fusion domain of the viral glycoprotein expressed on the surface of infected cells [2, 16] . several studies of different coronaviruses have shown that proteolytic cleavage of the s protein enhances viral infectivity. for mhv, separation of the s1 and s2 subunits enhances the fusion activity of the s2 subunit and increases viral infectivity [5, 32] . mutations that alter the furin protease recognition sequence (rxr/kr) located at the junction of the s1 and s2 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 [4, 9, 14] . conversely, the addition of trypsin to the culture medium can enhance the fusion of mhv-infected cells [13] . in contrast to mhv, the s protein of sars-cov does not show any evidence of proteolytic maturation to cleaved s1 and s2 subunits in mature virions [35] . instead, proteolytic cleavage of the s protein on the surface of infected cells occurs by exogenous proteases, mediating cell-cell fusion [25, 29] . similar to sars-cov, the s protein in most group 1 coronaviruses also does not exhibit cleaved s1 and s2 subunits during virus maturation and biogenesis [34] . several studies on tgev and pedv have used trypsinsupplemented culture media to induce cpe by syncytium formation in st cells and vero cells, respectively [15, 31] . furthermore, in the case of pedv, trypsin facilitates successful propagation in vero cells as well as other primate cell lines [15, 20] . however, the role of cellular and exogenous proteases on the cell entry of group 1 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. 3, 4 and 5, enhancement of virus penetration and cellcell fusion induced by the addition of trypsin suggests that the pedv s protein may also be cleaved into s1 and s2 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 220 kda in the absence of trypsin, while the protein was detected as two fragments of 140 kda and 125 kda in the presence of trypsin (fig. 6) . 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 s1 and s2 subunits [11] . 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 10 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 [11, 15] . 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. 3 , 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 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with clinically approved antiviral drugs date: 2004-04-17 journal: emerg infect dis doi: 10.3201/eid1004.030458 sha: doc_id: 309934 cord_uid: kcyao9i9 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 19 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-1b, α-n1, α-n3, and human leukocyte interferon α. these findings support clinical testing of approved interferons for the treatment of sars. s evere acute respiratory syndrome (sars) (1, 2) is an infectious disease caused by a newly identified human coronavirus (sars-cov) (3, 4) . the disease can produce severe pneumonia with a reported fatal outcome of 15% to 20%. currently, no effective drug exists to treat sars-cov infection (5) . the urgency of the outbreak has led to the empiric use of broad-spectrum antibiotics and antiviral agents in affected patients in several countries (6) (7) (8) (9) (10) (11) (12) . 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 (6) (7) (8) (9) (10) (11) (12) , oseltamivir (8) (9) (10) , foscarnet (8) , intravenous immunoglobulin (8) , 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 (5) . 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 (5) . 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 (13) : 1) direct antiviral effects, 2) inhibition of viral entry and replication at the cellular level by targeting virus-related processes, and 3) enhancement of host immune response. a total of 19 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 e6 cells to screen these antiviral compounds. sars-cov has been shown to infect vero e6 cells, an african green monkey kidney cell line (3) , 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 50% effective concentration (ec 50 ) 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 50 ranges from 1 to 100 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 (2a, 2b, n1, and n3, human leukocyte) and β (1a and 1b) were used. amantadine, an old antiviral compound, was also studied. different terms have been used to express antiviral activity, namely, ec 50 , 95% effective concentration (ec 95 ), and 50% inhibitory concentration (ic 50 ); table 1 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 3,000 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 1%. for plaque assays, fivefold drug dilutions were prepared by using growth media as specified below. vero e6 cells (american type culture collection, manassas, va) were propagated in 75 cm 2 cell culture flasks in growth medium consisting of medium 199 (sigma, st louis, mo) supplemented with 10% fetal calf serum (fcs; biological industries, kibbutz beit haemek, israel). sars-cov 2003va2774 (an isolate from a sars patient in singapore), which has been previously sequenced (14) , was propagated in vero e6 cells. briefly, 2 ml of stock virus was added to a confluent monolayer of vero e6 cells and incubated at 37°c in 5% co 2 for 1 h; 13 ml of medium 199 supplemented with 5% fcs was then added. the cultures were incubated at 37°c in 5% co 2 , and the supernatant was harvested after 48 h; in >75% of cultures, inhibition of cpe (3+) in each well was observed with an inverted microscope. the supernatant was clarified at 2,500 rpm and then divided into aliquots, placed in cryovials, and stored at -80°c until use. all virus culture and assays were carried out in the biosafety level-3 laboratory at the environmental health institute, according to the conditions set out in biosafety in microbiological and biomedical laboratories (15) . virus titer in the frozen culture supernatant was determined by using a plaque assay. briefly, 100 µl of virus in 10-fold serial dilution was added, in duplicates, to a monolayer of vero e6 cells in a 24-well plate. after 1 h of incubation at 37°c in 5% co 2 , the viral inoculum was aspirated, and 1 ml of carboxymethylcellulose overlay with medium 199, supplemented with 5% fcs, was added to each well. after 4 days of incubation, the cells were fixed with 10% formalin and stained with 2% 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. (16) , and all drugs were tested in quadruplicate. briefly, 100 µl of serial 10-fold dilutions of the drugs were incubated with 100 µl of vero e6 cells, giving a final cell count of 20,000 cells per well in a 96-well plate. the incubation period was 1 h at 37°c in 5% co 2 , except for the interferons, which were incubated overnight with the cells. ten microlites of virus at a concentration of 10,000 pfu/well was then added to each of the test wells. the plates were incubated at 37°c in 5% co 2 for 3 days and observed daily for cpe. the end point was the drug dilution that inhibited 100% of the cpe (cia 100 ) in quadruplicate wells. to determine cytotoxicity, 100 µl of serial 10-fold dilutions of the drugs was incubated with 100 µl of vero e6 cells, giving a final cell count of 20,000 cells per well in a 96-well plate, without viral challenge. the plates were then incubated at 37°c in 5% co 2 for 3 days and examined for toxicity effects by using an inverted microscope. trypsinized vero e6 cells were resuspended in growth medium and preincubated with interferons (serial fivefold dilution) in quadruplicate wells in 24-well plates. the next day, the medium was aspirated, and 100 µl of virus was added to each well at a titer of 100 pfu/well. after incubation for 1 h, the virus inoculum was aspirated, and a carboxymethylcellulose overlay containing maintenance medium and the appropriate interferon concentration was added. after 4 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 50% of plaques in each well (ic 50 ) was deter-mined. results were plotted in microsoft excel, and a polynomial of order three was used to approximate the data and extrapolate ic 50 and ic 95 values. high titers of infectious sars-cov, originally derived from a respiratory sample of a sars patient, were propagated on vero e6 cells. the cpe of sars-cov on vero e6 was evident within 24 hours after infection (figure 1 ). sars-cov-infected cells display a cpe characterized by the appearance of rounded cells and the destruction of the monolayer. a collection of 19 antiviral drugs was tested in the sars-cov cpe inhibition assay ( table 2) . 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 10 4 pfu/well and a high multiplicity of infection (moi = 0.5) rate were used. complete inhibition, expressed as cia 100 , was observed for interferon β-1b (betaferon) at 5,000 iu/ml, interferon α-n3 (alferon) at 5,000 iu/ml, interferon α-n1 (wellferon) at 250,000 iu/ml, and human leukocyte interferon α (multiferon) at 500,000 iu/ml. ribavirin also completely inhibited the cpe at 5,000 µg/ml ( table 3) . none of the other drugs showed complete inhibition of cpe, even at the highest concentration of drug tested (table 2) . rebif (ifn-β-1a) showed slight inhibition of cpe at 250,000 iu/ml, but the inhibition was not complete at the screening virus load of 10,000 pfu/well. likewise, roferon (ifn-α-2a) showed slight, incomplete inhibition at 50,000 iu/ml. because the criteria for ascertaining anti-sars-cov activity in this screen were set at 100% 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, 10 3 and 10 2 pfu/ well. all four drugs again showed inhibitory effect, although the cia 100 were dependent on viral loads (table 3) . at the lowest viral load the cia 100 were 5 iu/ml for both interferon β-1b (betaferon) and human leukocyte interferon α (multiferon); and 50 and 250 iu/ml for interferon α-n3 (alferon) and interferon α-n1 (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, 0.5-5 mg/ml. at high concentrations of ribavirin (0.2-1 mg/ml) cytotoxic effects were observed on veroe6 cells, as has been reported for other cell types (17, 18) . as such, we consider ribavirin to be inactive against sars-cov. a plaque reduction assay format with 100 pfu of sars-cov (moi = 0.0005) was conducted to determine the ic 50 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-n1 (wellferon), as production of this drug has been discontinued. cells were preincubated for 15 h with fivefold dilutions of drug. viral-induced plaques, which developed in 3 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 2 ). the ic 50 and ic 95 were determined to be 0.2 and 8 iu/ml for betaferon, 0.8 and 200 iu/ml for alferon, and 2 and 44 iu/ml for multiferon. betaferon, alferon, multiferon, wellferon, and ribavirin inhibited cpe in sars-cov-infected vero e6 cells, in decreasing order of potency. ribavirin, a drug widely used in initial efforts to manage sars infections, inhibited cpe completely at 500-5,000 µg/ml at virus loads of 100-10,000 pfu per well. the concentration range observed is much higher than concentrations that inhibit other viruses (respiratory syncytial virus, ed 50 2-8 µg/ml, hiv or resistant strains of rhinovirus, 50-100 µg/ml), including viruses that were tested on vero cells (west nile virus, new york isolate 178 µg/ml, and uganda isolate 41 µg/ml) (19) . in addition, the cpe 10,000 500,000 10,000 10,000 1,000 10,000 5,000 1,000 1,000 100 1,000 500 10 100 (17, 18) . 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 (20) (21) (22) . additionally, we have tested the effect of ribavirin and betaferon in combination (range of concentration of ribavirin, 1-100 µg/ml; range of concentration of betaferon, 0.1-10 iu/ml). at 1,000 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 (23) (24) (25) . the antiviral activity of interferons is mediated by direct effects on infected cells or by modulating an immune response (26) . interferons interact with specific surface cell receptors, leading to production of interferon-stimulated gene products such as 2′5′-oligoadenylate synthase and protein kinase pkr (27) . 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 3 to 5 million iu three times a week to 5 million iu daily. for interferon β, data regarding efficacy in the treatment of hepatitis c are conflicting, and interferon β (at doses of 3 to 6 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 2′5′ oligoadenylate synthase. interferon activity varies among different cell types (28, 29) , 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-1b, α-n1, α-n3, and human leukocyte interferon α exhibit antiviral activity in an in acute respiratory syndrome in chinaupdate 3: disease outbreak reported: geneva: the organization a major outbreak of severe acute respiratory syndrome in hong kong a novel coronavirus associated with severe acute respiratory syndrome a cluster of cases of severe acute respiratory syndrome in hong kong severe acute respiratory syndrome (sars) and coronavirus testing-united states clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study outbreak of severe acute respiratory syndrome in hong kong special administrative region: case report lung pathology of fatal severe acute respiratory syndrome identification of severe acute respiratory syndrome in canada identification of a novel coronavirus in patients with severe acute respiratory syndrome icu management of severe acute respiratory syndrome clinical features and short-term outcomes of 144 patients with sars in the greater toronto area antiviral drugs comparative full length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection biosafety in microbiological and biomedical laboratories initial in vitro screening of drug candidates for their potential antiviral activities in vitro effect of 1-beta-d-ribofuranosyl-1,2,4-triazole-3-carboxamide (virazole, icn-1229) on deoxy-ribonucleic acid and ribonucleic acid viruses an evaluation of a new antiviral agent 'virazole' against influenza virus infections identification of active antiviral compounds against a new york isolate of west nile virus interferon alpha2b alone or in combination with ribavirin for the treatment of relapse of chronic hepatitis randomised trial for interferon alpha2b plus ribavirin interferon alpha2b alone or in combination with ribavirin as initial treatment for hepatitis c prevention of experimental coronavirus colds with intranasal alpha-2b interferon intranasal interferon as protection against experimental respiratory coronavirus infection in volunteers the efficacy and tolerance of intranasal interferons: studies at the common cold unit biological properties of recombinant alpha-interferons: 40th anniversary of the discovery of 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-265263-r9e6bop3 authors: kassaa, imad al; hober, didier; hamze, monzer; caloone, delphine; dewilde, anny; chihib, nour-eddine; drider, djamel title: vaginal lactobacillusgasseri cmul57 can inhibit herpes simplex type 2 but not coxsackievirus b4e2 date: 2015-03-10 journal: arch microbiol doi: 10.1007/s00203-015-1101-8 sha: doc_id: 265263 cord_uid: r9e6bop3 this study aimed at demonstrating the antiviral activity of lactobacillus gasseri cmul57 (l. gasseri cmul57), l. acidophilus cmul67 and l. plantarum cmul140 against herpes simplex type 2 (hsv-2) and coxsackievirus b4e2 (cvb4e2), which are enveloped and naked viruses, respectively. these lactobacilli were non-cytotoxic and were able to reduce the cytopathic effect induced by hsv-2 in vero cell monolayers. however, lactobacilli were not active against cvb4e2. tested lactobacilli displayed anti-hsv-2 activity when they were co-incubated with the virus prior to inoculating the mixture to vero cell monolayers. the detection of hsv-2 dna by pcr in pellets of bacteria/virus mixtures let us to hypothesize that anti-hsv-2 activity of lactobacilli resulted from the viruses’ entrapment. this study showed the capabilities of vaginal lactobacilli to inhibit enveloped viruses such as hsv-2. activity of probiotic strains. related to this, botić et al. (2007) 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. 2012) . 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 2013) . 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. 2013) . 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. 2014a, 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. 2014b ). related to this, lactic acid, hydrogen peroxide and bacteriocins constitute the main anti-pathogen arsenal produced by vaginal lactobacilli (al kassaa et al. 2014a) . conti et al. reported that the anti-hsv-2 activity of l. brevis was originated by a molecule other than lactic acid and h 2 o 2 (conti et al. 2009 ). mastromarino et al. (2011) showed that another strain of l. brevis exerted anti-hsv-2 activity by a heat-resistant and non-protein cell surface component. subsequent to these findings (mastromarino et al. 2011) , khani et al. (2012) showed that l. rhamnosus enhanced macrophage viability in elimination of hsv-1, while zabihoullahi et al. (2012) reported that supernatants from different lactobacilli inhibited hsv replication. this study aimed at highlighting the anti-hsv-2 potential of vaginal lactobacilli recently isolated from a cohort of lebanese women. this report focuses, especially on l. gasseri cmul57, which is resulting hereafter as able to inactivate the enveloped hsv-2 but not the naked enterovirus cvb4e2. 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 cmul57, l. acidophilus cmul67 and l. plantarum cmul140 (al kassaa et al. 2014a ). these strains were grown in mrs broth (de man et al. 1960) and incubated for 24 h at 37 °c, under anaerobic conditions using anaeropack (biomérieux, france). the cells were harvested (1500 g, 15 min, 4 °c), and the pellets were washed twice with phosphate buffer solution (pbs) (ph 8.0). the number of cells was adjusted to 10 8 cfu/ml by using the turbidity (600 nm) and counting method on mrs (de man et al. 1960 ) agar plate. staphylococcus aureus (s. aureus) strain was grown in brain heart infusion (biokar, france) at 37 °c for 18 h. the number of colonies was determined on baird-parker agar (biokar, france). the vero cells (kidney epithelial from african green monkey) (atcc ccl-81) were grown in dulbecco's modified eagle's medium (dmem) (gibco, usa), supplemented with 10 % foetal calf serum (gibco, usa), and 1 % l-glutamine (gibco, usa), and incubated at 37 °c in the presence of 5 % co 2 in tissue culture flasks to a confluency of approximately 80 %. further, 2.5 × 10 4 cells/ml were seeded in 96-well culture cell microplates and incubated under the same conditions to 80 % confluency. the cell viability test was carried out using a commercialized kit "uptiblue" (uptima, interchim, france). briefly, the vero cells were seeded at 2.5 × 10 4 cells/well on 96-well microtitre plates and cultured until reaching 80 % 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 37 °c with 5 % co 2 for 2 h to insure the bacterial adhesion. after bacterial adhesion, the vero cell monolayers were washed three times with pbs followed by addition of 100 µl of uptiblue reagent. this mixture was incubated for 4 h at 37 °c with 5 % co 2 . absorbance was measured on an eia reader (lab systems; mtx labs, vienna, va, usa) at a test wavelength of 570 nm and a reference wavelength of 595 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 4, 24, 32 and 48 h of bacterial adhesion by using the uptiblue kit. therefore, vero cells at 80 % of confluency were incubated with 100 µl of tenfold serial dilutions (10 8 cfu/ml to 10 4 cfu/ml) and cultured up to 48 h before measuring cell viability. in addition, the presence of bacteria was measured as previously described (jacobsen et al. 1999) . briefly, a cell suspension containing 10 5 cells in 4-ml complete dmem medium was transferred to each well of a six-well tissue culture plate. when the cells reached 80 % confluency, the medium was removed 24 h prior to the adhesion assay and the cells were supplemented with dmem, without antibiotics. the monolayer cells were washed twice with 3 ml of pbs. an aliquot of 2 ml of dmem (without serum and antibiotics) was added to each well and incubated for 30 min at 37 °c. bacterial cells, at 10 8 cfu/ml, were resuspended in 1 ml of dmem medium (without serum and antibiotics) and added to different wells. the plates were incubated for 2 h at 37 °c in the presence of 5 % co 2 . the monolayer cells were washed five times with sterile pbs, and the adhesion score was determined by counting the adhered bacteria within 20 random microscopic fields after giemsa staining (jacobsen et al. 1999) . bacteria were grouped into three categories: nonadhesive (≤40 bacteria), adhesive (41-100 bacteria) and highly adhesive (>100 bacteria). the percentages of adhesion were determined by plating mrs agar plate followed by colony counting after 48 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. 1999 ). hsv-2 was isolated at the laboratory of virology (chru lille) from a patient with vaginal infection and was cultured on vero cell line. cvb4e2 is member of the human enterovirus b species of the enterovirus genus that can be grown on hep2 cell line and on vero cell line as well (sane et al. 2013) . 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 50 (50 % tissue culture infectious dose) titration method (landry et al. 2002) . the plaque reduction assay was carried out as previously described (zabihollahi et al. 2012 ) with some modifications. briefly, the vero cells were placed into 24-well culture plates (nunc, denmark) at a concentration of 1.7 × 10 5 cells per well and incubated for 24 h (in antibiotic free medium) until 98 % of confluency. the vero cells were inoculated with 100 pfu hsv-2 or 100 tcid 50 /ml cvb4e2 and incubated for 4 h. afterwards, the cell monolayers were washed and were overlaid with dmem, supplemented with 1.5 % (w/v) agarose (invitrogen, uk), 5 % fbs, 100 iu/ml penicillin and 100 μg/ml streptomycin (gibco, usa). after 72 h of incubation at 37 °c in 5 % co 2 atmosphere, the overlay medium was removed and the vero cell monolayers washed twice with pbs, followed by fixing with pure methanol for 30 min. counts of the viral plaques was done after staining with 0.5 % crystal violet (landry et al. 2002) . the dna extraction was carried out using the qiaamp minelute virus spin kit (qiagen, germany). the primers and probes targeting hsv-2 glycoprotein b were designed using the primer express 2.0 software (applied biosystems, usa). the primer sequences were fhsv-2 5′-cgcacctgcgggaaatc-3, rhsv-2 5′-gcgggcacacgtaaa-3′ and taqman probe sequence (hsv-2 probe): (vic) 5′-aggtcgaga acgcc-3′ (mgb). the qpcr was realized on abi 7500 (applied biosystems, usa) machine using the following pcr program: 2 min/50 °c (1 cycle), 10 min at 95 °c (1 cycle) and 45 cycles of (15 s/95 °c, 1 min/60 °c). taqman universal master mix (2×) (applied biosystems, usa) and 0.04 μm of primers and probe was used in the qpcrs. dna extracted from hsv-2 reference strain atcc vr-734d was used as control of qpcr. the viability of vero cells decreased to 81, 88, 91 and 25 %, after 24 h of incubation in the presence of 10 7 cfu/ml of l. gasseri cmul57, l. acidophilus cmul67 or l. plantarum cmul140 and s. aureus atcc33862, respectively. when the concentration of lactobacilli increased up to 10 8 cfu/ml after 24 h of incubation, the viability of vero cells was 85, 83 and 78 % for cmul57, cmul67 and cmul140, respectively. however, after 48 h of incubation, the cell viability diminished to 65 and 17 %, in the presence of lactobacilli strains and s. aureus atcc33862, respectively (fig. 1) . 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 2 h of incubation and reached about 80 % for lactobacilli, and 34 % for s. aureus atcc33862 ( table 1 ). the addition of lactobacilli strains to vero cell monolayers followed by their challenge with 100 pfu/ml hsv-2 reduced the cell death by 28, 16 and 17 %, respectively, compared with the control that contained vero cells without bacteria and challenged with hsv-2 (fig. 2) . the results of 2-h lactobacilli contact were quietly different to those obtained after 24-h contact (fig. 2) . there was no significant reduction in vero cells' death when they were challenged with 100 tcid 50 /ml cvb4e2 (fig. 2) . no impact on viability of vero cells was observed when l. gasseri cmul57 was replaced either by l. acidophilus cmul67 or by l. plantarum cmul140 (fig. 2) . however, when vero cells were infected with hsv-2 or cvb4e2 and then challenged with lactobacilli at 10 7 cfu/ml, the observed cytotoxicity remained unchanged (data not shown). the co-incubation of l. gasseri cmul57, l. acidophilus cmul67 or l. plantarum cmul140 strain with hsv-2 on vero cell monolayers has increased the cells viability by 65, 35 and 15 %, respectively, as compared to the control with 22 % viability, after exposure to the virus (fig. 3a) . in case of l. gasseri cmul57, the increase in post-treatment vero cell viability was lactobacilli cell concentration dependent (fig. 3b) . when the vero cell monolayers were inoculated with cvb4e2 at 100 tcid 50 /ml and lactobacilli at 10 7 cfu/ml, the cvb4e2-induced cytopathic effect was not modified. inhibition of the hsv-2-induced cytopathic effect by l. gasseri cmul57 was accompanied by a reduction in viral progeny as compared to the control (10 2 tcid 50 /ml vs. 10 5 tcid 50 /ml p = 0.02). however, there was no reduction in viral progeny with strains l. acidophilus cmul67 (10 4 tcid 50 /ml; p = 0.15) and l. plantarum cmul140 (1.5 10 4 tcid 50 /ml; p = 0.2) (fig. 4) . in case of l. gasseri cmul57 (fig. 5a) , the dmem supernatant (ph 4.8), neutralized supernatant (ph 6.9), and supernatant treated with protease and with catalase to degrade proteins and eliminate possible activity due to the presence of h 2 o 2 caused 0.4, 0.1, 0.14 and 0.21 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 cmul57-, l. acidophilus cmul67-and l. plantarum cmul140-derived neutralized supernatants enhanced slightly the viability of vero cell monolayer inoculated with 100 pfu hsv-2 with about 9.5, 5.4 and 2.4 %, respectively, due to reduction in viral infectious particles. these data are normalized to hsv-2 alone used as a control (table 2 ). this very weak inhibition prompted us to investigate whether the anti-hsv-2 activity of lactobacilli was exerted through an interaction between bacteria and hsv-2 particles. thus, l. gasseri cmul57 at 10 8 cfu/ml or dulbecco's modified eagle's medium (dmem, negative control) were incubated for 2 h at 37 °c in co 2 atmosphere in the presence of hsv-2 (10 4 pfu/ml) or cvb4e2 (10 3 tcid 50 /ml). afterwards, supernatant was collected by centrifugation (20,000 g, 3 min, 4 °c) and assayed on vero cells. after the treatment, the number of infectious hsv-2 aureus) atcc33862 at 10 7 cfu/ml. the cell viability was measured using uptiblue, and the results were expressed as the percentage of viability compared to the negative control (100 % viable vero cell monolayers). the results were the means (±sd) of at least three independent experiments particles was significantly reduced (15,000 vs. 100,000 pfu/ml p = 0.01), whereas that of cvb4e2 remained unchanged as compared to the control (9.8 10 2 tcid 50 /ml) (fig. 5b, 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 cmul57. thereof, the cycle threshold of the control assay was 26.2 ± 0.8 for 1000 pfu, and that of the pellet was 24.2 ± 0.9 (fig. 5d ). in contrast, the detection of hsv-2 dna in the final washing supernatant was almost negative (fig. 5d) . the bacterial pellet suspected to be containing hsv-2 infectious particles, as well as the washing supernatants were serially diluted (10 −1 to 10 −5 ) and added to vero cell monolayers. afterwards, a pra method was carried out to examine these samples' infectivity, by incubating them for 5 days at 37 °c in 5 % co 2 . after the incubation, no viral infection was observed for the supernatant of the final washing and for the bacterial pellet (fig. 5e ). vaginal lactobacilli play crucial role in prevention of urogenital infections such as bacterial vaginosis, vaginal candidiasis and viral infections such as those of hsv-2 (conti et al. 2009 ). hsv-2 is one of the most common sexually hsv-2 and cvb4e2 were used to challenge vero cells and then incubated for 6 h. the unbound virus particles were removed by three washings. results were recorded after 48 h of incubation and represented by the virus titration and expressed as logtcid 50 /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. 2013) . here, we demonstrate the anti-hsv-2 potential of l. gasseri cmul57, l. acidophilus cmul67 and l. plantarum cmul140 that were recently isolated from human vagina and characterized for their large antagonism and probiotic characteristics (al kassaa et al. 2014a ). the highest anti-hsv-2 activity was observed for l. gasseri cmul57. recently, zabihoullahi et al. (2012) showed the anti-hsv-2 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-2. further, the absence of cytotoxicity of lab probiotics is a prerequisite before undertaking any antiviral study. l. gasseri cmul57, l. acidophilus cmul67 and l. plantarum cmul140 used in this study are shown to be not cytotoxic for vero cells. safety of bifidobacterium adolescentis spm 0214 was established on vero cells , and that of e. faecium on porcine macrophage cell line strain and hsv-2 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-2 only, whereas the test wells refer to the wells containing hsv-2 co-incubated with cmul57 strain two main pathways (al kassaa et al. 2014b ). 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. 2009 ). here, this possibility is discarded because the assays conducted with the supernatants gathered from l. gasseri cmul57, l. acidophilus cmul67 and l. plantarum cmul140 cultures revealed either slight or absence of anti-hsv-2 activity. however, the anti-hsv-2 activity attributed to molecules of a other than h 2 o 2 and lactic acid bacteria was reported for l. brevis cd2, l. salivarius fv2 and l. plantarum fv9 isolated from human vagina (conti et al. 2009 ). 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. 1997) . 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. 2014b) . the data obtained here agree with this concept, as we show that l. gasseri cmul57 strain is active against enveloped virus (hsv-2), but not against naked one (cbv4e2). interestingly, co-incubation of hsv-2 and l. gasseri cmul57 experiments as well as the qpcr one let us to think that hsv-2 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. (1997) showed the capabilities of lactobacilli to trap human immunodeficiency virus (hiv) by binding the mannose sugar-rich "dome" of their attachment glycoprotein gp120. ivec et al. (2007) reported the trapping and adsorption of vesicular stomatitis virus (vsv) by lactobacillus and bifidobacterium species. lai et al. (2009) 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. (2013) 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. 2013 ) and poultry industry (seo et al. 2012) because viral diseases cause an enormous loss in the production in shrimp farms (lai et al. 2009 ), and avian influenza virus occurrence is responsible of severe losses to the poultry industry (seo et al. 2012) . overall, this study shows that hsv-2 envelope is a key element in the inactivation by vaginal l. gasseri cmul57. after the in vivo studies have been established, l. gasseri cmul57 could be recommended as vaginal probiotic to preventing hsv-2 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 0214 against herpes simplex virus type 1 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 2 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 asp2151 (amenamevir) herpes simplex virus 2: a boon to develop other sexually transmitted infections inhibition of herpes simplex virus type 2 by vaginal lactobacilli a medium for the cultivation of lactobacilli antiviral activity of trappin-2 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 1 human immunodeficiency virus type 1 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 2: role of cell wall associated components coxsackievirus b4 can infect human pancreas ductal cells and persist in ductal-like cell cultures which results in inhibition of pdx1 expression and disturbed formation of islet-like cell aggregates evaluation of leuconostoc mesenteroides yml003 as a probiotic against low-pathogenic avian influenza (h9n2) 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 1 university (france). the authors declare that there is no conflict of interest. key: cord-309469-2naxn580 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: 2019-01-05 journal: virology doi: 10.1016/j.virol.2018.12.019 sha: doc_id: 309469 cord_uid: 2naxn580 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 563 nucleotides excluding a poly(a) tail, is mainly derived from the 3′-untranslated region of ibv genome, and contains a 63-nt-long of terminal leader sequence derived from the 5′ 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: (1) viral ncrnas transcribed by rna polymerase (pol) iii. for example, two virus-associated (va) rnas encoded by human adenovirus (reich et al., 1966; steitz et al., 2010) , eber1 and eber2 encoded by epstein-barr virus (skalsky and cullen, 2015) , mirna precursors encoded by murine γ-herpesviral trna-pre-mirna chimeras (bogerd et al., 2010; bowden et al., 1997; diebel et al., 2010) and retrovirus (kincaid et al., 2012) , and intragenic viral small ncrna encoded by human bocavirus 1 (wang et al., 2017) ; (2) viral ncrnas transcribed by rna polymerase ii. for example, polyadenylated nuclear (pan) rna encoded by kaposi's sarcoma-associated herpesvirus (sun et al., 1996; zhong and ganem, 1997) , the~2-kb latency-associated transcript (lat) expressed by herpes simplex virus (bloom, 2004 ), a con-served~5-kb intron expressed by human cytomegalovirus (hcmv) (kulesza and shenk, 2004) , and a 7.2-kb rna expressed by mouse cmv (kulesza and shenk, 2006) , u-rich rnas (hsurs) produced by herpesvirus saimiri (hvs), (albrecht and fleckenstein, 1992; ensser and fleckenstein, 2005) ; (3) subgenomic ncrnas from single-stranded rna viruses by incomplete degradation of genomic rna by the cellular 5-3′ exonuclease xrn1. 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, 2014; roby et al., 2014; pijlman et al., 2008; akiyama et al., 2016) , 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., 2016a) . 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, 1991; wilson et al., 2014) . eber2 regulates the expression of a subset of ebv latent genes throung the interaction of eber2 and a cellular transcription factor paired box protein 5 (pax5) (arvey et al., 2012; lee et al., 2015) . degradation of mir-27 by mediated hsur 1 promotes activation and presumably proliferation of hvs-infected host t cells guo et al., 2014) . kshv pan rna is essential for virion production (borah et al., 2011) .β-herpesvirus hcmvencoded β2.7 prevents mitochondria-induced apoptosis, enabling steady atp production for viral processes and persistent infection (campbell et al., 2008; stern-ginossar et al., 2012) . sfrna produced by flaviviruses is required for cytopathicity and pathogenicity (pijlman et al., 2008) . it has been demonstrated to 1) interfere with cellular rna decay pathways by inhibiting xrn1 (moon et al., 2012) , 2) dampen the antiviral activity of type i interferon (schuessler et al., 2012) and 3) inhibit the rnai pathway in both the vertebrate and arthropod hosts, most likely by serving as a decoy substrate for dicer (schnettler et al., 2012) . inhibition of the host interferon response appears to be, at least in some flaviviruses, achieved by binding and inactivating cellular https://doi.org/10.1016/j.virol.2018.12.019 received 28 september 2018; received in revised form 19 december 2018; accepted 26 december 2018 regulators of translation of interferon-upregulated mrnas (bidet et al., 2014) . 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., 2016b) . 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, 2005) . like other coronaviruses, ibv can produce sgrnas via a discontinuous transcription mechanism to encode its structural proteins and specific accessory proteins (masters, 2006; sawicki et al., 2007) . briefly, ibv produces six mrna species in the infected cells, including its genomic mrna1, sgrna2 encoding spike (s) protein, sgrna3 encoding 3a, 3b and envelope (e) protein, sgrna4 encoding membrane (m) protein, sgrna5 encoding 5a and 5b, and sgrna6 encoding nucleocapsid (n) protein. recently, a low-abundance sgrna located between the sgrna4 and 5 has been identified (bentley et al., 2013) . in this study, we identify firstly an ncrna in the ibv-infected cells. moreover, we prove that this ncrna is derived mainly from 3′ utr of viral genome and is generated by discontinuous transcription process. vero and chicken embryo fibroblast df1 cells were maintained in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum (fbs), penicillin (100 units/ml) and streptomycin (100 units/ml) (invitrogen). a recombinant ibv (ribv) (fang et al., 2007) generated from an infectious clone reference genome (ibv beaudette p65, genbank accession number dq001339.1) 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 −80°c in 0.5-1 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., 2007) 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 t7 kit (ambion, austin, tx) according to the manufacturer's instructions, and electroporated into vero cells with one pulse at 450 v and 50 µf with a bio-rad gene pulser ii electroporator. the transfected vero cells were cultured overnight in 1% 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 6-well plates were infected with a dilution series of viruses for 1 h, washed twice with medium. cells were overlaid with 0.4% agar in fbs-free dmem, incubated at 37 ℃ for 3-4 days, fixed with 10% formaldehyde, and stained with 0.2% 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., 2009) . the allantoic fluid and different organs were harvested after the embryos were chilled at 4°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™ 1000 spectrophotometer (nanodrop technologies, inc., thermo fisher scientific, usa). reverse transcription (rt) was performed with oligo(dt)18 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)18 was used for cdna synthesis; for negative-stranded sgrna, ibv-5′end-f (5′-1 acttaagatagatattaatatata) was used. ibv-5′end-f and ibv-3′end-r (5′-27608 tgctctaactctatactagc) were used for pcr. ibv-infected cells at different time point post-infection were washed with pbs and lysed with 2 ×sds loading buffer containing 100 mm dithiothreitol (dtt), boiled at 100°c for 5 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 4°c or for 2 h at room temperature in blocking buffer (5% fat-free milk powder in phosphate-buffered saline (pbs) buffer containing 0.1% tween 20 (pbst)) and then was incubated with diluted primary antibodies in blocking buffer for 2 h at room temperature. after the membrane was washed three times with pbst, it was incubated with 1:2000 diluted anti-mouse or anti-rabbit igg antibodies conjugated with horseradish peroxidase (dako) in blocking buffer for 1 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 (2 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 7500 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 = 1). all experiments were performed in triplicate. in the virus-infected cells, ibv produces six mrna species, including the genome-length mrna1 and five subgenomic mrnas via a discontinuous transcription mechanism. this mechanism is mediated by transcription-regulating sequences (trss) in the 3′ 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 64 nucleotides derived from the 5′-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 mrna6 by rt-pcr, an unexpected pcr product was concurrently amplified in the ibv-infected vero cells as well as in chicken embryos (fig. 1) . it is smaller than mrna6. subsequently, this product was cloned and sequenced. sequence analysis indicated that it consists of 563 nucleotides excluding a poly(a) tail, is mainly derived from the 3′-utr (from nucleotides 27104-27608) of ibv genome, and contains a 63-nt-long of terminal leader sequence derived from the 5′ end of the viral genome (fig. 2) . 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 27149 and 27150 nt of ibv 3′-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. 3) , implying that the ncrna is an mrna. however, this virus was unstable and a deletion of 377 nucleotide acids (from 81 to 457 nt) of egep sequences was detected in passage 3 in vero cells (data not shown). further sequence comparison revealed that a sequence motif (uaaca), located in the junction between the 5′ end leader sequence and the 3′-utr of ncrna, is shared by 5′-terminus of 3′-utr and the core sequence (cuuaacaa) within ibv trs (fig. 2) . 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. 4 , compared to wild-type ribv, single mutation u27104a and a27108u had no or minor effect on ncrna synthesis; a27105u resulted in a significant reduction in the ncrna generation, while mutation c27107g, a27106u and deletion of five nucleotides (δ27104-08) completely abolished the synthesis of both positive-stranded and negative-stranded ncrna (fig. 4) , suggesting that at least three fig. 1 . 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)18 as a primer. pcr was performed using primers ibv-5′end-f and ibv-3′end-r. the amplicons were analyzed on 1% agarose gel electrophoresis. fig. 2 . sequence of the junction between ibv 3′-utr and the leader at 5′-end of viral genome, indicating the formation of a novel sgrna mediated by 27104 uaaca 27108 . vero cells were infected with ribv at an moi of 1 pfu/cell. total rna extracted from the infected cells was used for reverse transcription using primer oligo(dt)18. pcr was carried out using primers ibv-5′end-f and ibv-3′end-r. pcr product was cloned and sequenced. nucleotides (a27105/a27106/c27107) 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., 2013) notably, the mutant virus carrying four mutations (a27100u/a27111u/g27113c/g27114c) was also unable to produce ncrna (fig. 4) , suggesting these nucleotides are required for ncrna production. because the sequence motif (a27100/a27111/ g27113/g27114) located downstream of the truncated cs (uaaca) also exists downstream of the cs (cuuaacaa) in the leader trs (table 1 , marked in bold), our result prove that the sequences downstream of the cs exert a stronger influence on coronavirus sgrna synthesis (sola et al., 2005) . taken together, the results confirm that the ncrna generation involves a discontinuous transcription process in which the 5′ leader sequence and 3′-utr are fused through the transcription-regulating sequences in the 3′ end of the leader and in the 5′ end of the 3′-utr. the mutant virus c27107g 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. 5a) . 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. 5b, 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 3′ end of the leader (trs-l) and in the intergenic region preceding each mrna body (trs-b) during negative rna synthesis (baric and yount, 2000; zúñiga et al., 2004; masters, 2006; sawicki et al., 2007) . the five sgrnas (mrnas 2-6) of ibv, which are readily detected by northern blotting, possess the canonical core sequence (5′-cu(u/g)aacaa-3′) ( table 1 ). 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, 2000; hussain et al., 2005; bentley et al., 2013) . in this report, we have identified the existence of a novel sgrna derived mainly from the 3′ utr of ibv in the ibv-infected cells (fig. 1) . the synthesis of this sgrna is mediated by a truncated cs ( 27104 uaaca 27108 ) identical to nucleotides of 3-7 of ibv cs. among which at least three nucleotides (a27105/a27106/c27107) are involved in the effective sgrna generation (figs. 2 and 4) , providing more evidence for the use of noncanonical transcriptional signals in synthesis of coronavirus sgrnas. in addition, we verified that the sequence motif (a27100/a27111/g27113/g27114) located downstream of the truncated cs (uaaca) is necessary for ncrna generation (fig. 4) , reinforcing the importance of nucleotides immediately flanking cs in coronavirus sgrna synthesis (sola et al., 2005) . interestingly, when blast search in genbank, we find that the sequence motif (uaaca) is conserved at 5′ end of 3′ utr of ibv strain beaudette and its derivants, arkansas dp1, and turkey coronavirus but not for strain m41, h120, h52, a2, 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. 5 . effect of ncrna on plaque morphology and viral replication. a. plaque assay. vero cells in 6-well plates were infected with a dilution series of ribv and mutant virus ibv-c27107g for 1 h, respectively. after washing twice with medium, cells were overlaid with 0.4% agar in fbs-free dmem, incubated at 37 ℃ for 3-4 days, fixed with 10% formaldehyde, and stained with 0.2% crystal violet. b. quantitative analysis of sgrna synthesis of n and s. total rna (2 μg) extracted from the vero cells infected with ribv and ibv-c27107g at an moi of 0.5 pfu/cell at 8 and 20 h post-infection was used for reverse transcription using primer oligo(dt)18 respectively. amplification and data collection were performed as manufacturer's instruction (applied biosystems 7500 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 = 1). all experiments were performed in triplicate. c. western blotting. vero cells were infected 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sarcoma-associated herpesvirus (human herpesvirus 8) 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. 31572490) and the department of science and technology, hubei provincial people's government, china (no. 2013bhe020) key: cord-287488-h102xn29 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-2 isolation from the first reported patients in brazil and establishment of a coordinated task network date: 2020-10-23 journal: mem inst oswaldo cruz doi: 10.1590/0074-02760200342 sha: doc_id: 287488 cord_uid: h102xn29 background: severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was confirmed in brazil in february 2020, 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-2 isolates from the first confirmed cases of coronavirus disease 2019 (covid-19) in brazil. methods: after diagnosis in patients that returned from italy to the são paulo city in late february by rt-pcr, sars-cov-2 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 (e6, ccl-81 and hslam), with clear cytopathic effects, and characterised by full genome sequencing, electron microscopy and in vitro replication properties. virus stocks viable (titre 2.11 × 10(6) tcid50/ml, titre 1.5 × 10(6) pfus/ml) and inactivated from isolate sars.cov2/sp02.2020.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. (1) however, in 2003 and 2012, respectively, the emergence of highly pathogenic severe acute respiratory syndrome (sars-cov) (2) and middle east respiratory syndrome (mers-cov) (3) revealed that this virus group may also cause severe respiratory illness in humans. in december 2019, in wuhan, china, a novel coronavirus, member of the β coronavirus family, has been identified as the source of a pneumonia outbreak (4) and this novel virus was named as severe acute respiratory syndrome coronavirus 2 (sars-cov-2), by the international committee on taxonomy of viruses (ictv). (5) in brazil, the four endemic covs circulate annually (6) (7) (8) (9) (10) (11) and the first case of coronavirus disease 2019 (covid19) was reported on february 26, 2020 (https://covid.saude. gov.br) when sars-cov-2 was detected in a 61-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, (12) there were 81,109 confirmed covid-19 cases in 38 countries. after these first reported patient, cases in brazil started to rise reaching 3,846,153 infected persons and 120,462 deaths on august 29 (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-2 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-2 from the first two patients diagnosed with the novel coronavirus disease in brazil. we describe its genomic sequence (sars-cov-2/sp02/human/2020/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 74683917.1.0000.5467). all specimens were handled under the laboratory biosafety guidance required for the novel coronavirus (2019-ncov) by the world health organization (who) (13) at bls3 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-19 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-19 in the são paulo city. the specimens were collected on day 2-4 post-symptom onset, placed in 1-2 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-2, the extraction of total nucleic acid (rna and dna) from the collected samples (200 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 (2019-ncov) by who (13) at bls3 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 7500 sds real-time pcr machine (applied biosystems, weiterstadt, germany), using a published protocol and sequence of primers and probe for e gene. (14) 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. (2) virus isolation -we used vero e6 cells for isolation and initial passages. we cultured vero e6 in dulbecco minimal essential medium (dmem) supplemented with 10% 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 25 cm 2 cell culture flask in a concentration of 5 × 10 5 cells/ml. after 24 h, we removed the culture medium, washed three times with fbs free-dmem and inoculated aliquots (500 μl) of the clinical specimens into the flask. after 1 h of incubation (adsorption), we completed the volume for 5 ml with dmem supplemented with 2.5% fbs and 1% of penicillin-streptomycin. we grew the inoculated cultures in a humidified 37°c incubator in an atmosphere of 5% co 2 and observed for cytopathic effects (cpe) daily up to 72 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 50 /ml) -vero e6 and ccl-81 cells were seeded into 96-well plate (5 × 10 4 cells/ml), 24 h before the experiment. virus was 10-fold serially diluted in medium (10 -1 to 10 -12 ). medium was removed from plates, virus dilutions applied in sextuplicate and incubated at 37°c. visualisations were performed daily in an inverted light microscope (axiovert 100, carl zeiss oberkochen) to observe the cpe. after 72 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 50 /ml and calculated using the spearman & kärber algorithm, as described by hierholzer & killington. (15) plaque forming units (pfu/ml) -virus titration was carried out in 24 wells plates seeded with vero e6 and ccl-81 cells at a concentration of 1 × 10 5 cells/well. after 24 h and a cell confluence of 80-90%, dilutions 10 -1 to 10 -10 in dmem 2.5% fbs of the virus was transferred in duplicate (100 µl/well) to the seeded plates. after 1 h adsorption at 37 o c 5% co 2 , the wells were completed with an overlay of carboxymethyl cellulose (cmc) with dmem, 2% fbs and 1% of penicillin-streptomycin, and plates incubated at 37 o c in 5% co 2 and stained with naphtol blue black dissolved in sodium acetate-acid acetic. plates were observed and stained from 48 to 96 h post-inoculation (h.p.i.). both virus titration (tcid 50 / ml and pfu/ml) were made after the third passage of the isolated virus (t2). samples were adsorbed to glow-discharged carbon filmcoated copper grids (400 mesh, cf400-cu, electron microscopy sciences). the grids were washed with ultrapure water treated with depc and negatively stained with uranyl acetate 2% (w/v) with blotting on filter paper after each step. a fei tecnai g20 200 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 (e6, ccl-81 and hslam) plus a human epithelial type 2 (hep-2) cells, at concentration of 5 × 10 4 cells/ml, were tested for the propagation of the sars-cov-2 by inoculation at a multiplicity of infection (moi) of 0.02. the culture medium consisted of dmem supplemented with 2.5% of fbs. aliquots of cell-associated and supernatants compartments were collected every 12 h up to 96 h.p.i. for virus quantification via tcid 50 / 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 8.1 (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. (16) 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 550 equipment (illumina) for sequencing with mid 300 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 hg19 reference genome, taxonomic identification of other pathogens and genome recovery through manual curing. quality control was performed using cutadapt (17) to filter sequences by length (< 50 bp), average quality (q p < 20) and trim options to remove low quality ends (9 bp to 5' end and 5 bp to 3' end). passed qc reads were mapped to hg19 human reference genome using bwa (18) mem with default parameters. not mapped reads were submitted to assembly using spades 1.13. (19) contigs were inspected and manually curated using geneious 2020.1 to generate a final assembly. complete genome was compared to sars-cov-2 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 1 (hiae01), a 61-years-old male patient, and patient 2 (hiae02), a 32-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 1 was initially diagnosed with a community-acquired pneumonia and received antimicrobial therapy. both were confirmed for covid-19 by hospital israelita albert einstein (hiae), in the são paulo city on february 26 (hiae01) and 28 (hiae02), 2020. a summary of clinical characteristics of the patients is described in table. lombardy is considered the centre of the covid-19 outbreak in italy (20) and has a high influence of the first wave of sars-cov-2 introduced in brazil. (21) virus isolation -before isolation in cell cultures, we tested the samples using a one-step multiplex rt-qpcr for the detection of 18 additional different respiratory viruses (22) 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 e6 cells. the initial sample collected from hiae01 was freezed and thawed before inoculation and no virus propagation was obtained. a second sample, from the same patient, was obtained "fresh" (conserved at 4ºc for no longer than 12 h) and we could successfully isolate sars-cov-2. sample from hiae02 was inoculated "fresh" from the first moment. the failure to isolate the virus from the first sample collected from hiae01 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. 1 . three days post infection, the isolation of sars-cov-2 from hiae02 was confirmed by rt-qpcr, electron microscopy and whole genome sequencing (fig. 1) . the cycle threshold (ct) value and genome copy numbers (rna copies/ml) of the pre-inoculated sample was ct 17.49 and 8.46 × 10 6 (fig. 2a) . rna quantification of passages 2 and 3 after 72 h.p.i. gave values of 10.12 -1.19 × 10 9 copies/ml and 9.22 -2.18 × 10 9 copies/ml, respectively. since virus isolation from hiae02 (sars.cov2/sp02.2020.hiae.br) was faster, all the subsequent studies were carried out with this isolate after passage 3. virus isolation (passage 0) from hiae01 (sars.cov2/sp01.2020.hiae.br) was confirmed by rt-qpcr (ct 15.02 -4.4 × 10 7 copies/ml) and whole genome sequencing, being stored at -80ºc. negative staining transmission electron microscopy of the sars.cov2/sp02.2020.hiae.br, here after referred as sp02/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 80 to 100 nm (fig. 2b ). the cpes were characteristic of sars-cov-2: cell rounding, detachment of the cell monolayer and formation of loose cells on the surface, for both vero e6 and ccl-81 cells and similar to previously observed effects. (23) (24) (25) (26) nonetheless, the cpes were more evident in ccl-81 cells. the virus isolate was titrated after two blind passages following isolation (t2) and the cpes were more clearly observed in ccl-81 cells for tcid 50 /ml (2.11 × 10 6 ) and pfus (1.5 × 10 6 ). for pfus titration, effects were not clear for vero e6 cells, and for vero ccl81, only a few small plaques were visible at 72 h.p.i., being much larger and more visible at 96 h.p.i. (supplementary data, fig. 1 ). hartcourt et al. (23) described effects more visible for vero e6 cells when compared to ccl-81. other successful sars-cov-2 isolation reports were based on vero e6, vero ccl-81 and vero hslam (23) (24) (25) (26) (27) showing that the three cell lines are permissive for sars-cov-2. virus growth kinetics in different cell lines -we examined sars-cov-2 growth kinetics in three vero cell lines (e6, ccl-81 and hslam) and compared with hep-2 cells. all cells were inoculated with the sars-cov-2 isolate (sp02/bra) and cultured under similar conditions. virus titre quantification analysis of cell-associated and supernatants compartments indicated that similar levels of infectious sars-cov-2 were produced in all three vero cell lines of vero, but not in hep-2 cells, that proved to be not permissive to virus replication. the peak of viral titre was detected 48 h.p.i. (10 7 tcid 50 /ml) after an initial eclipse phase. cpes was not observed until 48 h.p.i. and reached a peak at 72 h.p.i. (supplementary data, fig. 2 ). 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 72 h.p.i. (fig. 3b) . rnacp:tcid 50 ratios did not differ significantly (p > 0.05) among the tested vero cell lines (fig. 3c ). in addition, these cell lines appeared to release few noninfectious particles at early time points of the infection. the rnacp:tcid 50 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-2 cells (fig. 3a , b). any clear-characteristic cpe of sars-cov-2 was observed in hep-2 cells (supplementary data, fig. 2) . similarly to other studies with sars-cov (28, 29) and sars-cov-2, (23, 26) 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-2 wuhan-hu-1 (nc_045512) and inmi 1/ita (mt066156) were compared with sp02/ bra directly isolated from patient's sample (mt126808) and after cultivation in vero cells (mt350282) using mafft multiple aligner tool (algorithm = auto and pam = 1). alignment shows only two mutations at the cultivated strain (> 99.993% similarity). mutations occurred at the nsp2 and spike proteins (fig. 4) . distribution network -until march 20, 2020, the sending of the material comprised 31 different research groups, in public and particular university/hospitals, at 10 different states in brazil (fig. 5) . 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 bsl3 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-2 and the development of methods and strategies for virus treatment and prevention. sarscov-2 isolates were set available to the scientific community by different groups in other countries. (23, 27) the first delivery to the research community at the são paulo state was set on march 13. the virus distribution by the brazilian mail company was first set on march 18, 2020 and, in less than 13 h, the biological material was delivered to rio de janeiro (222.26 miles away from the são paulo city), the state of minas gerais (304.28 miles) and the state of rio grande do sul (815.94 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-2 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-2 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-19 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-2/sp02/human/2020/bra from de clinical sample and sars-cov-2/human/bra/ sp02cc/2020 from cell culture isolation have been deposited in the genbank (accession mt126808 and mt350282, 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 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metagenomic identification of viral pathogens in clinical samples by 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-19 epidemic in italy: effects of emergency containment measures routes for covid-19 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 2 from patient with 2019 novel coronavirus disease, united states virus isolation from the first patient with sars-cov-2 in korea serological and molecular findings during sars-cov-2 infection: the first case study in finland comparative tropism, replication kinetics, and cell damage profiling of sars-cov-2 and sars-cov with implications for clinical manifestations, transmissibility, and laboratory studies of covid-19: an observational study isolation and rapid sharing of 2019 novel coronavirus (sars-cov-2) from the first patient diagnosed with covid-19 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-19 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-330772-i7cfmw9x 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: 2019-12-03 journal: amb express doi: 10.1186/s13568-019-0916-0 sha: doc_id: 330772 cord_uid: i7cfmw9x 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 2013, outbreaks of new variants of porcine epidemic diarrhea virus (pedv) have caused high mortality and morbidity in piglets, leading to great economic losses (lee 2015) . 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 2015; song and park 2012) . this highly contagious disease has spread quickly in porcine industries in several countries (jung and saif 2015; lee 2015) . 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. 2015; deping song et al. 2017) . 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. 2017) . 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. 2018; lin et al. 2019) . 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 2019) . it has been demonstrated that b. licheniformis exhibiting antimicrobial activity against pathogens might be due to the production of antibacterial biosurfactants (lin et al. 2019) . 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. 2019 ). 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 c1008 cells (american type culture collection (atcc) no. crl-1586) were maintained in dmem (gibco, ny, usa) supplemented with 10% fetal bovine serum (hyclone, utah, usa) and 1% penicillin-streptomycin-amphotericin b (gibco, ny, usa). the passage 6 pedv-pintung 52 strain (pedvpt-p6) viral stock was used at a titer of 1 × 10 5 tcid 50 /ml. air-dried solid-state fermentative cultivates of b. licheniformis (weigmann) chester (atcc ® 27811 ™ ) were suspended in distilled water and heated at 30 °c for 30 min. supernatants were harvested after centrifugation at 13,000 rpm for 30 min. after adjusting the ph value to 2.0 with 6 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 1998) . briefly, after filtration, samples were subjected to analysis via spd-10a hplc (shimadzu, japan) with a pre-packed lichrospher 100 rp-18 column (merck, darmstadt, germany). the mobile phase was a mixture of 3.8 mm trifluoroacetic acid and 200 ml ddw with 800 ml 100% methanol. elution was performed at a flow rate of 1 ml/min and determined with a uv detector (10a vp, shimadzu, tokyo, japan) at 210 nm. the gradient strategy was as follows: 0-3.5 min, 60% a to 93% a; 3.5-20 min, keeping 93% a and 7% b (a, acetonitrile; b, ultrapure water); min pressure 0 bar, max pressure 400 bar, pressure stability 10 bar; injection volume 10 μl; syringe speed 8 μl/s; flush volume 800 μl. liquid chromatography-mass spectrometry (lc-ms) full scan positive mode was performed with m/z ranging from 200 to 2000. fifteen 4-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: (1) control (n = 5); (2) pedv (n = 5); and (3) pedv + blfp (n = 5). these pigs were fed a commercial diet mixed with or without 5 kg/l blfp as feed additives for 7 days prior to the viral challenge, and were challenged with or without 5 × 10 5 tcid 50 of the virulent pedvpt-p6 (p6) at 5 weeks of age (table 1) . 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 4-scale: 0, normal feces; 1, pasty; 2, semi-fluid; 3, fluid. all piglets were weighed weekly and sacrificed 19 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, ntu105-el-00087). real-time quantitative rt-pcr (qpcr) was performed based on a previously described method with modification (chang et al. 2017) . the viral rna was extracted from 200 μl of culture supernatants using cador ® pathogen 96 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 60 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 19 dpi to evaluate the safety of blfp in pigs. representative tissue samples were collected, fixed in 10% neutral-buffered formalin, processed routinely, sliced into 5-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 96-well microplate (corning life sciences, corning, ny, usa) at a density of 20,000 cells per well 1 day prior to the experiment. after removing the culture supernatant, ten-fold serially diluted blfp crude extract in pi medium (dmem supplemented with 0.3% tryptose phosphate broth (sigma-aldrich, mo, usa), 0.02% yeast extract (acumedia, michigan, usa), and 10 μg/ml of trypsin (sigma-aldrich, mo, usa) was added to the cell monolayer. after 48 h, the culture supernatant was removed and 100 μl alamarblue ™ was added (g-biosciences, st. louis, usa; 10% in pbs). after 2 h of incubation at 37 °c, the plate was read with the excitation wavelength at 575 nm and with the emission wavelength at 590 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 g1, which consisted of vero cells without any treatment, was included to represent 100% reduction of alamarblue. all data were further calculated as a percentage of the untreated group g1. 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 50% cytotoxicity concentration (cc 50 , the concentration of blfpat which cellular viability was reduced to 50%) 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 96-well microplate (20,000 cells/well) 1 day before the experiment. blfp crude extract at 150 ppmin pi medium were added to each well in different orders to treat 200 tcid 50 /ml pedv infections in cells. the experiment included five groups (as illustrated in fig. 4a to further investigate the antiviral mechanism in the post-drug group (g6), the replication kinetics of pedv in vero cells with or without crude extract of blfp were compared. vero cells were seeded in a 96-well microplate (20,000 cells/well) 1 day before the experiment. the crude extract of blfp at 150 ppm in pi medium was added to 200 tcid 50 /ml pedv-infected cells. culture supernatants and cells were collected separately at the 2, 8, 12, 24, and 48 h post-infection. viral titers in culture supernatants were determined using the reed-müench method (reed 1938) and are expressed as the 50% tcid 50 /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 10-fold dilution and the subsequent 10-fold serial dilution of the supernatants were added to 20,000 cells/well vero cells on 96-well microplates. after 1 h of incubation, the inoculants were aspirated, and the cells were washed with pi medium two times and then cultured in pi medium. after 72 h, the plate was subjected to cytopathic effect (cpe) observation and the titer of pedv was determined using the reed-müench method (reed 1938) and is expressed as the 50% tcid 50 /ml. to determine the ic 50 , vero cells were seeded in 96-well microplates (20,000 cells/well) one day before the experiment. ten-fold serially diluted crude extract of blfp in pi medium was added to vero cells 1 h after 200 50% tissue culture infectious dose (tcid 50 ) pedv infection. after 48 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 50 . to elucidate the direct virucidal activity of crude extract of blfp against pedv, the pedvpt-p6 viral stock at a titer of 1 × 10 5 tcid 50 /ml was mixed with or without 150 ppm blfp for 1 h at 4 °c. the mixtures were serially diluted 10-fold in pi medium and added to vero cells. after 72 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 < 0.05 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 < 0.05 considered significant. all data were analyzed using graphpad prism software (graphpad prism inc.) as shown in fig. 1 , hplc analysis of the b. licheniformis extract showed a single peak (fig. 1b) at the retention time of 16-17 min, which is identical to the standard substance surfactin derived from bacillus subtilis (fig. 1a) . (fig. 1) . to assess the efficacy of the antiviral ability of blfp against pedv, clinical symptoms after viral challenge were recorded daily (fig. 2) . the fecal conditions of pigs in all groups were scored as normal (score = 0) 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 (5/5) exhibited normal clinical signs (score = 0) 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 2 days post infection (dpi). two of these five pigs (2/5) exhibited semi-fluid feces (score = 2) at 2 dpi, while 3/5 pigs showed typical semi-fluid (score = 2) to watery diarrhea (score = 3) at 2 to 8 dpi, and eventually recovered after 8 dpi. in the pedv + blfp group, most of the pigs exhibited pasty feces (score = 1) at 2-5 dpi. only one pig exhibited transient typical watery diarrhea (score = 3) at 6 dpi. compared to the pedv-infected group with control food, the severity and duration of typical diarrhea (scores 2-3) were reduced in the pedv + blfp group. to quantify pedv-associated fecal viral shedding to evaluate the antiviral efficacy of blfp against pedvpt-p6, rt-qpcr was used to detect fecal viral shedding. in the pedv group (the grey line in fig. 3) , fecal viral shedding was first detected (3.92 ± 3.88 log 10 ge) at 2 dpi, found to gradually increase to peak viral load (5.71 ± 3.38 log 10 ge) at 5 dpi, and was continuously detected until 12 dpi. in the pedv + blfp group (the black line in fig. 3) , 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. 4) . a b fig. 2 clinical scoring of fecal consistency. compared to pedv-infected groups, the appearance and duration of typical diarrhea (score 2-3) 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 3 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 5 kg/ton are safe in conventional pigs up to 26 days of feeding. to determine the median cytotoxic concentration (cc 50 ) of blfp crude extract in vero cells, the blfp crude extract was serially diluted 10-fold from 150 to 0.015 ppm and added to cells. 48 h post-incubation (hpi), no cytotoxicity due to blfp crude extract was observed in vero cells (fig. 5) . to determine the maximal inhibitory concentration, the post-drug group g6 was used to evaluate the maximal inhibitory effect of blfp crude extract by cocultivating fig. 3 fecal shedding of pedvpt-p6 detected in piglets fed < 5 kg/ton blfp in each group (n = 5). 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 log10 values ± sd. the viral rna loads of inoculated groups and the control group were compared using a non-parametrical kruskal-wallis test fig. 4 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 50 value of 0.07 ± 0.45 ppm (fig. 6) . to elucidate the antiviral activity of blfp crude extract against pedv, 150 ppm of blfp crude extract was added to the culture at different time points during the viral infection. as shown in fig. 6a , 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 g3-g5-cells treated with blfp crude extract 1 h before infection, at the same time as infection, and 1 h after viral infection, then replaced with fresh pi medium-were calculated as 50.7 ± 16.2%, 26.0 ± 20.1%, and 44.3 ± 5.8%, respectively, and showed no significant dose-dependent effect of blfp crude extract against pedv. the half-maximal inhibitory concentration (ic 50 ) of blfp crude extract against pedv in vero cells is 0.07 ± 0.45 ppm. data are presented as the mean ± sd out of three test replicates differences from those in the pedv-infected group g2 (fig. 7b) . for the post-drug group g6, significant inhibition of pedv-induced cpe, with an 81.5 ± 7.61% reduction of alamarblue staining, was observed compared to that in g2 and other groups (g3-g5). similarly, the amount of viral rna in supernatants from g6, ranging from 6.26 to 7.39 log 10 ge, was significantly lower than that of g2-g5, ranging from 8.41 to 9.26 log 10 ge (fig. 7c) . additionally, the viral titer of supernatants in the g6 group, which was less than 10 tcid 50 /ml, was lower than that of g2-g5, ranging from 1 × 10 4 to 1 × 10 5 tcid 50 /ml. moreover, when evaluating the direct virucidal activity of blfp crude extract against pedv, a reduction of the viral titer of pedv from 10 5 to 10 4 tcid 50 /ml was observed. to further investigate the antiviral mechanism in the post-drug group (g6), 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 1.78 × 10 5 at 24 hpi to 3.16 × 10 6 tcid 50 /ml at 48 hpi (fig. 8a) , 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 24 and 48 hpi (fig. 8b) . however, no significant differences in intracellular viral titers (fig. 8c ) or intracellular viral rna (fig. 5d) were noted in pedv-infected cells treated with or without blfp crude extract. since late 2013, 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 2011; kwon et al. 2013) and in vivo (cho et al. 2012; kim et al. 2015) . herein, we demonstrated that: (1) blfp crude extract presents no cell toxicity to vero cells, and exhibits significant antiviral ability against pedvpt-p6 in vero cells when biosurfactants are co-cultivated with pedv; (2) piglets supplemented with blfp at concentrations of 5 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. 2006 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. 1997; wang et al. 2017) . lipopeptide biosurfactants have been previously suggested to possess probiotic attributes for animal and human use (schneider 1998; sen 2010) . 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. 1997 ) 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. 2008 ) and weaned piglets (torres et al. 2017) 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. 8 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 50% tcid 50 /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 < 0.05) 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 5 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 500 kg/ton leads to necrosis of hepatocytes in rats (hwang et al. 2009 ). 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 5 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 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epithelial cells evaluation of genetic and developmental toxicity of surfactin c from bacillus subtilis bc1212 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, ntu105-el-00087). not applicable. the authors declare that they have no competing interests. key: cord-253616-7jyui5ca authors: lai, zheng-zong; ho, yi-jung; lu, jeng-wei title: harringtonine inhibits zika virus infection through multiple mechanisms date: 2020-09-07 journal: molecules doi: 10.3390/molecules25184082 sha: doc_id: 253616 cord_uid: 7jyui5ca mosquito-borne zika virus (zikv) is a flavivirus that came under intense study from 2014 to 2016 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 (rp9-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 10,700 nucleotides in length and encodes approximately 3400 amino acids, which translate a single polyprotein. the polyprotein produces three structural proteins (capsid, pre-membrane, and envelope) and seven nonstructural proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5) via viral and cellular proteolysis [1, 2] . among these viral proteins, the envelope protein is responsible for viral entry and influences host attachment [3] . however, the nonstructural proteins are related to viral rna replication and virion assembly [4] . zikv was first discovered in rhesus macaques in ugandan forests in 1947. the first outbreak of zikv occurred in 2007 on yap island in the western pacific ocean, followed by a large epidemic in central and south american countries in 2015-2016 [5] . 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 96-well microplates; cytotoxicity was assessed for 48 h using a cck-8 assay, which evaluated cell proliferation by measuring cellular metabolic activity. the cell viability of vero cells was approximately 83 or 90% at harringtonine concentrations up to 1250 or 625 nm after 48 h treatment, respectively ( figure 1a ). to avoid drug-induced cell cytotoxicity of harringtonine treatment, this was limited to 650 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 = 0.01 under different concentrations of harringtonine for 48 h. for this, vero cells monolayer were cultured in 12-well microplates overnight, infected with 1000 ffu zikv per well, and incubated with raising concentrations of harringtonine from 156, 312 and 625 nm for 48 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 1b-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 1e ,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 48 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 < 0.05; ** p < 0.01; *** p < 0.001. scale bar: 50 μm. to further evaluate the individual stages of the virus life cycle, a time of addition assay was performed ( figure 2a ). harringtonine (625 nm) was administered at different stages of zikv infection with moi = 0.1, and then the levels of zikv rna in cells, as well as virus titers in supernatants, were determined after 24 h treatment. the zikv group and full-treatment group were, respectively, negative control and positive control. both levels of zikv rna production ( figure 2b ) and progeny yield ( figure 2c ) reduced at all processes of the virus life cycle. the strongest inhibition effect of harringtonine was observed at the post-treatment stage (approximately 3 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 = 0.1 for 2 h absorption. then, harringtonine 625 nm was added at 0, 6, and 12 h after the inoculum removed. the viral rna level was detected after 24 h incubation. based on the above data, we have determined that harringtonine effectively interferes at late stage of zikv infection ( figure 2d ). the viability was determined with a cell counting kit 8 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 48 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 < 0.05; ** p < 0.01; *** p < 0.001. scale bar: 50 µm. to further evaluate the individual stages of the virus life cycle, a time of addition assay was performed ( figure 2a ). harringtonine (625 nm) was administered at different stages of zikv infection with moi = 0.1, and then the levels of zikv rna in cells, as well as virus titers in supernatants, were determined after 24 h treatment. the zikv group and full-treatment group were, respectively, negative control and positive control. both levels of zikv rna production ( figure 2b ) and progeny yield ( figure 2c ) reduced at all processes of the virus life cycle. the strongest inhibition effect of harringtonine was observed at the post-treatment stage (approximately 3 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 = 0.1 for 2 h absorption. then, harringtonine 625 nm was added at 0, 6, and 12 h after the inoculum removed. the viral rna level was detected after 24 h incubation. based on the above data, we have determined that harringtonine effectively interferes at late stage of zikv infection ( figure 2d ). . 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 625 nm of harringtonine was added at 0, 6, and 12 hpi (hour post infection). at 24 hpi, the viral rna level was analyzed by rt-qpcr (d). statistical significance was analyzed from t-test compared with the zikv group: * p < 0.05; ** p < 0.01; *** p < 0.001. statistical significance was analyzed from t-test compared with the zikv group: * p < 0.05; ** p < 0.01; *** p < 0.001. our previous studies have shown that virus could bind onto cells, but could not enter into the cells at 4 • c. thus, 4°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 625 nm harringtonine was added during zikv infection (moi = 0.1) at 4°c, then the treated cells were washed, and fresh medium was added at 37°c. after 24 h, rna and virus titer were detected and used to verify the effect of viral binding. furthermore, the cells were infected with zikv (moi = 0.1) at 4 • c for 1h incubation, then the inoculum was removed, washed to replace with 625 nm harringtonine, and incubated at 37 • c for another hour. subsequently, 625 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 = 0.1 through virus binding ( figure 3a ,b) and virus entry processes ( figure 3c,d) . besides, the cells were infected with zikv (moi = 0.1) at 4 • c for 1 h, and then incubated at 37 • 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 3e ,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 3h incubation to verify the virucidal ability. then, the zikv supernatant was progressed 10-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 312 and 614 nm ( figure 3g ). we produced a timeline of time of binding, entry, replication, and release assays ( figure 3h ). interestingly, the antiviral effect of harringtonine was also observed even at the pre-treatment stage, when cells were pre-treated with harringtonine for 3 h before virus infection (figure 2a,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 5978 ( figure 4a,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 < 0.05; ** p < 0.01. to further assess the antiviral activity of harringtonine against other virus, the japanese encephalitis reporter virus (rp9-gfp strain) with moi = 0.01 was used. briefly, vero cells were seeded in 12-microplates overnight. cells were then infected with 1000 ffu rp9-gfp virus in the presence of harringtonine at different concentrations for 48 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 156 to 625 nm harringtonine exhibited a dose-dependent anti-rp9-gfp activity ( figure 5a-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 (rp9-gfp strain) with moi = 0.01 was used. briefly, vero cells were seeded in 12-microplates overnight. cells were then infected with 1000 ffu rp9-gfp virus in the presence of harringtonine at different concentrations for 48 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 156 to 625 nm harringtonine exhibited a dose-dependent anti-rp9-gfp activity ( figure 5a-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 2014 to 2016. 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 [17, 18] . previous research on the natural cephalotaxus alkaloids harringtonine, homoharringtonine, and cephalotaxine majorly showed antitumor effects, such as antileukemic activity [11, 19, 20] . cephalotaxus alkaloid antitumor effects were suggested via their inhibitory effects on protein synthesis and partly on dna synthesis [21, 22] . 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-2 [12, 13, [23] [24] [25] [26] [27] [28] . 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 [29] . the concentration of harringtonine used in this anti-zikv study was no more than 625 nm to avoid drug-induced cytotoxicity and kept the cell viability of vero cells remained above 90% ( figure 1a ). this concentration of harringtonine was much lower than that used in the treatment of chikv with 10 µm harringtonine in bhk21 cells [13] . 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 2) . 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. [13] . denv-infected cells revealed the increase of subpolysomal mrnas which might correlate to the repression of translation at the initiation stage [30] . harringtonine, through a block following 60s 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 [30] . other studies reported that harringtonine inhibited protein synthesis by blocking poly(u)-directed polyphenylalanine synthesis and peptide bond formation [21] , and interfered with large ribosome subunit [13] . 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 [21, 31] . 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 [13, 32] . 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 3a ,c) and virion progeny ( figure 3b,d) . harringtonine also revealed the virucidal ability which could destroy the virion stability ( figure 3g ). 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 [33] [34] [35] . 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 50 and cc 50 of harringtonine are 287.94 nm and >10 µm, while the ec 50 and cc 50 of cephalotaxine are 40 µm and >300 µm. the selectivity index (si) of harringtonine is 34.73, while the si of cephalotaxine is 7.5. 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 40 µm cephalotaxine reduced the infection ability of zikv by about 70%, but did not show more effects at a concentration of 80 µm (data not shown) [16] . however, the harringtonine has a better inhibitory effect (64.5% at 312 nm and 93.8% at 625 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 4 ). 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 [22] . in this study, the anti-jev effects of harringtonine was also investigated and demonstrated that harringtonine possessed dose-dependent antiviral activities ( figure 5a -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-81) was used in this study, as it is more permissive to zikv (pravabc59; genbank sequence accession: ku501215) replication; vero cells were cultured in dulbecco's modified eagle medium (dmem) supplemented with 5% fetal bovine serum (fbs) and antibiotics under a 5% co 2 incubator at 37 • c. harringtonine was purchased from chemfaces (catalog number: cfn90891), dissolved in 10% dimethyl sulfoxide (dmso) as a stock of 2 mm, and stored at −20 • c until use. green fluorescence protein-expressing japanese encephalitis reporter virus (rp9-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 c6/36 mosquito cells (c6/36; atcc, crl-1660) and vero cells, respectively. cytotoxicity of harringtonine was determined using the cell counting kit 8 (cck-8, dojindo laboratories, kumamoto, japan). increasing concentrations of harringtonine with fresh medium were added to cells in 96-well microplates in triplicates and incubated at 37 • c in a 5% co 2 incubator. after a 24 or 48 h incubation period, the medium was replaced with 100 µl of fresh medium containing 10 µl of cck-8 reagent for 1 h. the absorbance at 450 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, tri200), after that rna levels were measured using the one-step 2× rt-qpcr mix sybr green kit (bioman; catalog number: qrp001). rt-qpcr was performed on a roche lightcycler 480 (roche applied science, indianapolis, in) at the following conditions: 42 • c, 20 min; 95 • c, 10 min; (95 • c, 10 s; 62 • c 15 s; 72 • c 20 s) for 40 cycles. the primers used to detect zikv, jev and β-actin (internal control) were as follows: zikv: forward primer 5'-ttggtcatgatactgctgatgc-30 and reverse primer 5'-ccttccacaaagtccctattgc-3', jev: forward primer 5'-tccgtcaccatgccagtctt-3' and reverse primer 5'-gaggatgattctgtaagtatctaggtatagagccc-3', and b-actin: forward primer 5'-aggcaccagggcgtgat-3' and reverse primer 5'-gcccacataggaatccttc tgac-3' [36] . data were analyzed using the 2 -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 2 h incubation time, and cells were overlaid under semisolid dmem containing 1.5% methylcellulose for 48 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 12-well plates were fixed with 4% formaldehyde at room temperature for 1 h and were then permeabilized with an equal ratio of chilled methanol and acetone (1:1) for 30 min. cells were then washed three times with phosphate-buffered saline (pbs) and blocked with 5% skimmed milk for 1 h and were stained with anti-flavivirus envelope protein 4g2 primary antibody (1:1000 dilution; produced in-house) for 2 h. subsequently, cells were washed three times with pbs again, and alexa fluor 488-conjugated goat anti-mouse igg secondary antibody (1:1000 dilution; jackson immunoresearch laboratories, inc.; west grove, pa, usa) was added at 25 • c for another 2 h incubation. stained cells were visualized using an inverted fluorescence microscope (olympus ckx41, olympus, japan), as previously reported [36] . total cell lysates were harvested by adding ripa lysis buffer supplemented with protease inhibitors. anti-flavivirus envelope protein 4g2 antibodies (1:1000 dilution; produced in-house) and rabbit anti-β-actin polyclonal antibodies (1:4000 dilution; finetest, wuhan, china, catalog number: fnab00869) 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 12-well microplates. drug-containing medium (625 nm of harringtonine) was added at different time points relative to the 2 h period of cell infection with approximately 5000 ffu of zikv (moi = 0.1). for the pre-treatment group (pre), harringtonine was added 3 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 2 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 24 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 [16, 37, 38] . for binding assay, zikv (moi = 0.1) were inoculated onto vero cell monolayers in culture medium with or without 625 nm of harringtonine for 1 h at 4 • c. cells were then washed twice with pbs, and fresh medium was added for 24 h incubation at 37 • c in a 5% co 2 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 = 0.1) was added to vero cells at 4 • c for 1 h. cells were then washed twice with pbs, and fresh medium with or without 625 nm of harringtonine was added for another 1 h incubation at 37 • c in a 5% co 2 incubator. thereafter, cells were washed twice with pbs again before being added to fresh medium. after a 24 h incubation period, the levels of intracellular viral rna and virus titers in supernatants were determined by rt-qpcr and ffa, respectively [36, 39] . the cells were infected with zikv (moi = 0.1) at 4 • c for 1 h, and then incubated at 37 • c for another hour. when zikv entered into the cell and added the specified concentration of harringtonine. after 1 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 [16, 39] . zikv (3 × 10 4 ffu) was respectively mixed with harringtonine at 0, 156, 312, and 625 nm at 37 • c for 3 h; virus titers were then determined by ffa [16] . the zikv envelope proteins (5ire) crystal structure was obtained from the protein data bank (pdb). three-dimensional ligand structures of (chemspider id: 23089589) was obtained from the chemspider database and converted into the pdb format using pymol software, as previously reported [36] . patchdock was used in this project to conduct molecular docking analyses. finally, the conformations were then ranked according to docking scores [40] . the data obtained from this study were statistically analyzed in triplicate and using graphpad prism 8.0 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 < 0.05 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 h9n2 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-2: a treatment option for covid-19 remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov-2 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 u2504 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 2020, 25, 4082 key: cord-010369-x9z8dg6a 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 huh7.5.1-8 and african green monkey kidney-derived vero date: 2020-04-24 journal: plos one doi: 10.1371/journal.pone.0232274 sha: doc_id: 10369 cord_uid: x9z8dg6a 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-7-derived cell clone, huh7.5.1–8, which is highly permissive to hepatitis c virus infection. here, we have characterized flavivirus infection in the huh7.5.1–8 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, huh7.5.1–8 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 (17d-204 strain). quantification of cellular and extracellular viral rna revealed that high jev production in huh7.5.1–8 cells can be attributed to rapid viral replication kinetics and efficient virus release early in infection. in a plaque assay, huh7.5.1–8 cells developed jev plaques more rapidly than vero cells. although this was not the case with yfv plaques, huh7.5.1–8 cells developed higher numbers of yfv plaques than vero cells. sequence analysis of cdna encoding an antiviral rna helicase, rig-i, showed that huh7.5.1–8 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 5/6−12, indicating functional loss of rig-i in the cells. these characteristics of the huh7.5.1–8 cell line are helpful for flavivirus detection, titration, and propagation. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the huh-7 (no. jcrb0403) and vero (no. jcrb9013) cell lines were purchased from the japanese collection of research bioresources (jcrb) cell bank, national institute of biomedical innovation (osaka, japan). huh7.5.1-8, huh7.5.1, and huh-7 cells were maintained at 37˚c in an atmosphere of 5% co 2 in dulbecco's modified eagle's medium (dmem; wako pure chemical industries, osaka, japan; no. 044-29765) supplemented with 10% (v/v) fetal bovine serum (fbs; sigma-aldrich, st. louis, mo, usa; no. 172012-500ml), 0.1 mm nonessential amino acids (nacalai tesque, kyoto, japan; no. 06344-56), 100 u/ml penicillin g, and 100 μg/ ml streptomycin sulfate (nacalai tesque; no. 26253-84). vero cells were maintained at 37˚c in an atmosphere of 5% co 2 in eagle's minimal essential medium (emem; wako pure chemical industries; no. 051-07615) supplemented with 5% (v/v) heat-inactivated fbs, 100 u/ml penicillin g, and 100 μg/ml streptomycin sulfate. for comparative analysis, huh7.5.1-8 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 (s1a fig). the jev nakayama strain (isolated in japan in 1935) and jev rat strain [21] were propagated in vero cells. the yfv 17d-204 strain, a current vaccine strain derived from the 17d strain [22] , 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 −80˚c and used as virus stocks. these virus strains were chosen because they could be used in our biosafety level 2 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 [23] with modifications. in brief, cell monolayers grown in 12-well plates (corning costar 1 ; no. 3513) were inoculated with virus samples in 0.4 ml per well of culture medium with reduced heat-inactivated fbs (2%) and were incubated at 37˚c for 2 h. after removal of the samples, the cells were incubated in 2 ml of overlay medium consisting of emem (nissui pharmaceuticals, tokyo, japan; no.05901), 0.22% (w/v) nahco 3 , 2% (v/v) heat-inactivated fbs, 2 mm l-glutamine (nacalai tesque; no. 16948-04), and 1% (w/v) methylcellulose (tokyo chemical industry, tokyo, japan; no. m0185) at 37˚c for 5 days. the cells were fixed with 10% (v/v) neutral buffered formalin (wako pure chemical industries; no. 133-10311) and stained with 0.0375% (w/v) methylene blue. wells with 10−100 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 2.5 ml per well of dmem (nissui pharmaceuticals; no. 05919) supplemented with 0.2% (w/v) nahco 3, 2% (v/v) heat-inactivated fbs, 2 mm l-alanyl-l-glutamine (nacalai tesque; no. 04260-64), 0.1 mm nonessential amino acids, and 1.25% (w/v) methylcellulose. jev plaque formation in vero cells was not so different between the dmem overlay medium and the emem overlay medium (s1b fig). after fixation and staining, plates were scanned with a document scanner, and image data were obtained (s11 fig). to improve plaque visibility, an invert filter was applied to the image data, and highlights were adjusted using photoshop elements (version 14; 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 001-2) and the viral nucleic acid extraction kit i (favorgen biotech; no. favnk 001-2), respectively, according to the manufacturer's instructions. to determine the copy numbers of jev rna (nakayama strain; genbank accession no. ef571853.1), the rna fraction was subjected to one-step qrt-pcr using the thunder-bird 1 probe one-step qrt-pcr kit (toyobo, osaka, japan; no. qrz-101) with primers #1 and 2 and a hydrolysis probe #3 (table 1 ). the following reactions were performed on the lightcycler 1 96 system (roche applied science): reverse transcription at 50˚c for 10 min and denaturation at 95˚c for 1 min, followed by 40 cycles of 95˚c for 15 s and 56˚c for 45 s. to normalize cellular jev rna levels, the copy numbers of mrna encoding glyceraldehyde-3-phosphate dehydrogenase (gapdh) (human, genbank accession no. nm_002046.3; chlorocebus sabaeus, genbank accession no. xm_007967342) in the same rna samples were determined with primers #6 and 7 and a hydrolysis probe #8 ( table 1 ; the sequences of #6−8 are homologous to both the human and monkey sequences) under the same qrt-pcr conditions, except that the annealing temperature was 60˚c. a standard curve was established using serial dilutions of plasmids encoding the jev e protein (bases 962 to 2491) 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 #4 (table 1) total rna from cells was reverse transcribed with an oligo dt primer using the primescript™ ii 1st strand cdna synthesis kit (described above). the first strand cdna was used as a template to amplify a cdna fragment encoding rig-i (ddx58) (genbank accession no. (table 1) . upon sequence determination, nucleotide variations were excluded that were not consistent with the rna-seq data from huh7.5.1 (drr018792) and huh7.5.1-8 (drr018793) [20] or the whole-genome sequence data from huh-7 and huh7.5.1-8 (ddbj dra database under the project id prjdb7928) [25] . all the experiments were done with three biological replicates and repeated as described in fig comparison of jev production between huh7.5.1-8, huh7. first, the huh7.5.1-8 cell line was compared with the parental huh7.5.1 and the ancestral huh-7 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 1−4 d pi. the relative jev titer of these cell lines increased and peaked at 2−3 d pi in a similar manner ( fig 1a) . under the same seeding conditions, doubling times of huh7.5.1-8, huh7.5.1, and huh-7 cells were not significantly different (after bonferroni correction, p > 0.0167) (s2b 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 1b) , 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. huh7.5.1-8 and huh7.5.1 cells similarly developed jev plaques, whereas huh-7 cells began to die at 3 d pi and detached from the surface of the well irrespective of infection at 5 d pi ( fig 1c) . these results indicated that the huh7.5.1-8 and huh7.5.1 cell lines are better than the huh-7 cell line for a jev plaque assay. although huh7.5.1-8 and huh7.5.1 cell lines were comparable in terms of jev infection, the huh7.5.1-8 cell line has the advantage of being phenotypically more stable than the huh7.5.1 cell line [20] . thus, we hereafter focused on the huh7.5.1-8 cell line and compared it with the vero cell line regarding jev production. jev production in huh7. for comparative analysis, we used the vero cell subline jcrb9013, in which jev production was almost equivalent to or slightly higher than that in other vero cell sublines (s4 fig). huh7.5.1-8 and vero cells under high and low confluency were infected with jev at moi 0.1, and then the virus titer in culture supernatant was monitored from 1 to 4 d pi (fig 2) . the culture supernatant of huh7.5.1-8 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 2a) compared with low-cell-density infection ( fig 2b) , implying that cell-cell contacts promote viral spread in huh7.5.1-8 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 (s2 fig). a parallel viability assay showed that huh7.5.1-8 cells lost viability faster than vero cells irrespective of cell density (fig 3) . the same trends were observed in jev production and cell viability at smaller moi (0.01), although high confluency appeared to be needed (s5 fig) . these results showed that jev production in the huh7.5.1-8 cell line is higher than that in the vero cell line during early infection times and that the huh7.5.1-8 cell line is more susceptible to jev-induced cell death than the vero cell line. the particle-to-pfu ratio was calculated for huh7.5.1-8-and vero-derived jev particles, and the ratio of the values of both particles was determined (fig 4) . both virus particles showed a similar particle-to-pfu ratio, ruling out the possibility that huh7.5.1-8-derived virus particles had a higher infectivity than vero-derived ones. the reason for large variations found at 2 and 3 d pi remains unknown, but that at 3 d pi may have been partly due to variable amounts of non-infectious viral rna leaking from dead huh7.5.1-8 cells (fig 3) . in a representative experiment (s6 fig) , the actual value of the particle-to-pfu ratio was minimally~600, which is roughly consistent with a previous report [26] . taken together with the results of fig 2, these results suggested that huh7.5.1-8 cells produce a higher amount of jev particles than vero cells during early infection times. next, we compared the jev replication kinetics between huh7.5.1-8 and vero cells by monitoring intracellular and extracellular viral rna levels during 0−73 h pi. an initial rise in intracellular viral rna level in huh7.5.1-8 cells was detected as early as 9 h pi, whereas the rise for vero cells was not remarkable during 0-12 h pi (fig 5a [a]) . similarly, the level of extracellular viral rna from huh7.5.1-8 cells rose earlier than that from vero cells (fig 5b [a] ). although the level of intracellular viral rna was not different between the two cell lines after 12 h pi (fig 5a [b] ), the level of extracellular viral rna was higher for huh7.5.1-8 cells than for vero cells during 12−48 h pi (fig 5b [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 2-fold higher in huh7.5.1-8 cells than in vero cells at 1 d pi (fig 6) . thereafter, the value for huh7.5.1-8 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,1) or an unpaired two-tailed t test using bonferroni correction and p values less than 0.0167 were considered statistically significant. ns, not significant. (c) cells were seeded at 4 x 10 5 cells per well of a 12-well plate one day before infection and then infected with jev at 40 pfu (determined with vero cells) per well. the cells were fixed and stained at 3−5 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 0.0167 were considered statistically significant. similar results were obtained in two other independent experiments (s3 fig). https://doi.org/10.1371/journal.pone.0232274.g001 huh7.5.1-8 and vero cells. thus, huh7.5.1-8 cells may release jev more efficiently than vero cells at least at 1 d pi. collectively, these results suggested that rapid viral replication kinetics and efficient early virus release contribute to high jev production in huh7.5.1-8 cells. jev plaque formation (size and number) were compared between huh7.5.1-8 and vero cell lines at 3−5 d pi. as shown in fig 7a and s7a fig, clearly visible plaques were developed in huh7.5.1-8 cells at as early as 3 d pi, but not in vero cells at that time. in the course of the comparison of flavivirus production between huh7.5.1-8 and vero cell lines infection, plaques in huh7.5.1-8 cells grew more rapidly than those in vero cells. similar results were observed when the cells were infected with another jev strain, rat (fig 7b and s7b fig) . these results showed that the huh7.5.1-8 cell line supports more rapid jev plaque formation than the vero cell line. the rapid plaque formation in huh7.5.1-8 cells appears to reflect their high jev productivity and susceptibility to jev-induced cell death. the plaque numbers of the nakayama strain at 5 d pi were comparable between the two cell lines (fig 7a and s7a fig), whereas those of the rat strain tended to be higher in the huh7.5.1-8 cell line than in the vero cell line (fig 7b and s7b fig) . these results indicated that the difference in plaque numbers between the two cell lines varies by each virus strain. fig 2, 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 huh7.5.1-8-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 (1 and 2 d pi) or seven (3 d pi) independent experiments (the reason for the different sample sizes was that one experiment lacked data for 3 d pi). statistical significance was determined by a one-sample t test. values in parentheses indicate p values, and those less than 0.05 were considered statistically significant. (fig 8c and 8d) . the combined data showed that virus yield in huh7.5.1-8 cells was significantly higher than that in vero cells for high confluency at 2 d pi and low confluency at 1 and 2 d pi. furthermore, huh7.5.1-8 cells were more susceptible to yfv-induced cell death than vero cells (fig 8e and 8f ), as observed with jev. these results suggested that the huh7.5.1-8 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 9 and s9 fig) . yfv plaques began to appear at 4 d pi in both cell lines. although plaques in huh7.5.1-8 cells were slightly smaller than those in vero cells, huh7.5.1-8 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 huh7.5.1-8 cells can be mainly attributed to their high infection efficiency. although yfv plaques developed in vero cells at 4 d pi in fig 9, no cell death was observed with these cells at this time point in fig 8e and 8f . a possible explanation for this discrepancy in cell death may be the difference in culture conditions: the serum concentration was 10% for fig 8 and 2% for fig 9. in addition, the medium in the case of jev, the lower the serum concentration is, the more cell death occurs [27] . recently, codon 55 in the rig-i gene from the huh7.5.1-8 cell line was found to be heterozygous: mutant-type ata (ile) and wild-type aca (thr) [25] . to confirm whether the huh7.5.1-8 cell line is defective in rig-i, we amplified the rig-i cdna derived from total rna isolated from huh-7, huh7.5.1 and huh7.5.1-8 cells. an amplicon corresponding to the expected size for the rig-i sequence (~3 kbp) and another, with a smaller size (~2 kbp), were detected in the three cell lines (fig 10a) and directly sequenced (fig 10b) . the 3-kbp and 2-kbp cdnas from the huh-7 cell line exclusively contained the wild-type codon 55. in contrast, the 3-kbp cdnas from the huh 7.5.1 and huh7.5.1-8 cell lines contained the mutanttype codon 55, whereas the 2-kbp cdnas from these cell lines contained the wild-type codon 55. cloning and sequencing of the cdnas of huh-7 and huh7.5.1-8 cell lines revealed that the 3-kbp cdnas were full-length (fl1, 2), whereas the 2-kbp cdnas lacked exons 6−12 (sv1 found in both cell lines) or 5−12 (sv2 found in huh7.5.1-8) (fig 10c and s10 fig) . rig-i protein isoforms deduced from the splice variants (sv1, 2) completely lacked a helicase atpbinding domain, indicating that they are defective like rig-i protein with a mutated atpbinding site [28] . these results were consistent with the above findings [25] and indicated that huh7.5.1-8 is a rig-i null mutant cell line. although the huh7.5.1-8 cell line is more permissive to hcv infection than the huh7.5.1 cell line [20] , we found that this was not the case with jev infection: the huh7.5.1-8 and the parental huh7.5.1 cell lines were comparable in terms of jev productivity, susceptibility to jev-induced cell death, and jev plaque formation (fig 1) . even the ancestral huh-7 cell line was comparable except for plaque formation. these findings indicate that genetic factors contributing to the high permissiveness of the huh7.5.1-8 cell line to hcv infection are specific rather than broadly proviral. one such genetic factor might be involved in stable hcv receptor expression [20] . unlike the huh7.5.1-8 and huh7.5.1 cell lines, the huh-7 cell line was not maintained under our culture conditions for the plaque assay (fig 1c) , likely because our huh-7 cells had a lower fitness to cell culture than huh7.5.1-8 and huh7.5.1 cells. here, we found three differences in outputs of flavivirus infection between huh7.5.1-8 and vero cell lines. first, huh7.5.1-8 cells produced higher amounts of infectious jev and yfv than vero cells early in infection (figs 2 and 8a-8d ). in the case of jev, the high virus productivity of huh7.5.1-8 cells may be attributed not to the increase in infectivity of a virus particle (fig 4) , but to viral rapid replication kinetics and efficient virus release early in infection (figs 5 and 6) . therefore, huh7.5.1-8 cells are more suitable than vero cells for obtaining large amounts of flavivirus in a short period of time. recently, huh-7 cells were shown to produce higher amounts of zika virus than other cells including vero e6 cells (atcc crl-1586) early in infection [29] . considering that jev production in huh7.5.1-8 cells and the ancestral huh-7 cells were comparable (fig 1a) , high virus productivity early in flavivirus infection may be a common feature among the huh-7 lineage. in addition, a similar trend was observed when huh-7 cells were infected with middle east respiratory syndrome coronavirus [30] . thus, the feature may not be limited to flavivirus infection. second, we found that huh7.5.1-8 cells were more susceptible to jev/yfv-induced cell death than vero cells (figs 3, 8e and 8f ). these characteristics of the huh7.5.1-8 cell line may facilitate cell viability-based screenings of antiviral host factors and agents for flaviviruses. meanwhile, the potent cell death observed in huh7.5.1-8 cells appears to be a part of the reason for plateaued or reduced virus production late in infection. therefore, genetic engineering of the huh7.5.1-8 cell line to make it less susceptible to virus-induced cell death might yield a cell line with more flavivirus production. fig 2. (a, b) culture supernatants were harvested at 23, 47, 74, and 98 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 (s8 fig). (c, d) the titer ratio was calculated by dividing the titer value of huh7.5.1-8 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 s8 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 0.05 were considered statistically significant. huh, huh7.5.1-8 cells. https://doi.org/10.1371/journal.pone.0232274.g008 comparison of flavivirus production between huh7.5.1-8 and vero cell lines third, we found that huh7.5.1-8 cells developed jev plaques rapidly (fig 7) and formed a larger number of yfv plaques (fig 9) compared with vero cells, though the difference in plaque numbers between the two cell lines varied by each virus. the jev plaque phenotype of huh7.5.1-8 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 huh7.5.1-8 cells can be utilized to shorten the period of plaque assay. in addition, the high-number plaque phenotype observed with yfv infection in huh7.5.1-8 cells can be utilized to improve the sensitivity of detection and titration of yfv by comparison of flavivirus production between huh7.5.1-8 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 huh7.5.1-8 cells. in addition, the higher tolerance of vero cells to flavivirus-induced cell death, compared with huh7.5.1-8 cells, may be a feature that makes the vero cell line advantageous for vaccine production. on top of these findings, we provided evidence that huh7.5.1-8 is a rig-i null mutant cell line. our results suggested that, in a huh7.5.1-8 cell, one allele of the rig-i gene generates full-length mrna with a t55i 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-7 cell line. codon 55 of rig-i gene of huh7.5.1 cells was heterozygous as was that of huh7.5.1-8 cells, showing that the heterozygosity of the codon was stable at least during the time span for isolation of the huh7.5.1-8 clone from huh7.5.1 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 [31] and mouse cell lines [32, 33] , jev production and susceptibility to jev-induced cell death were not dramatically altered in the huh7.5.1-8 and huh7.5.1 cell lines compared with the huh-7 cell line (fig 1a and 1b) , implying that the rig-i pathway may not work or may be counteracted by viral mechanisms [34] under our experimental conditions. in conclusion, our study highlighted the characteristics of the huh7.5.1-8 cell line that are helpful for improvement of flavivirus propagation, detection, and titration. further study is needed to investigate the potential versatility of the huh7. 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 0.05 were considered statistically significant. huh, huh7. (sv1, 2) . alignment of nucleotide sequences was performed using genetyx version 14.0.0 (genetyx co., tokyo, japan). (pdf) s11 fig. unprocessed image data, related to figs 1c, 7, 9 , s1b, s3, s7 and s9. (pdf) s1 raw images. raw image data (fig 10a) . (pdf) estimated global incidence of japanese encephalitis: a systematic review fact sheets yellow fever studies on sv40 in tissue culture: preliminary step for cancer reserach in vitro studies on sv40 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 1-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, sa14-14-2 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 trim25-mediated antiviral signal transduction viral rna detection by rig-i-like receptors rig-i in rna virus recognition isolation and characterization of an huh.7.5.1-derived cell clone highly permissive to hepatitis c virus characterization of the e-138 (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 huh7 and huh7.5.1-8 cell lines development of multiplex real-time reverse transcriptase pcr assays for detecting eight medically important flaviviruses in mosquitoes flavivirus activates phosphatidylinositol 3-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 lgp2 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 mda5 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 tlr3 and rig-i in mediating the inflammatory response in mouse microglia following japanese encephalitis virus infection japanese encephalitis virus ns5 inhibits type i interferon (ifn) production by blocking the nuclear translocation of ifn regulatory factor 3 and nf-kap-pab we thank dr. francis v. chisari (the scripps research institute, la jolla, ca, usa) for providing the huh7.5.1 cell line. we also thank dr. tomohiko takasaki (department of virology i, national institute of infectious diseases) for providing the yfv 17d-204 strain. key: cord-313596-kc8loqyj 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: 2014-09-28 journal: dna res doi: 10.1093/dnares/dsu029 sha: doc_id: 313596 cord_uid: kc8loqyj 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 25,877 putative protein-coding genes were identified in the 2.97-gb genome sequence. a homozygous ∼9-mb deletion on chromosome 12 caused the loss of the type i interferon gene cluster and cyclin-dependent kinase inhibitor genes in vero cells. in addition, an ∼59-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 27 march 1962 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. 1, 2 vero cells were found to be highly susceptible to various types of viruses including simian polyoma virus sv-40, 1,2 measles virus, 3 rubella virus, 4,5 arboviruses, 6, 7 and adenoviruses 7 soon after their establishment, and were later found to be also susceptible to bacterial toxins including the diphtheria toxin, 8 heatlabile enterotoxins, 9 and shiga-like toxins (or 'vero' toxins). 10, 11 after their global distribution, 12,13 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 14 and are non-tumourigenic when a cell passage was not prolonged. 15, 16 therefore, the vero cell lineage has been successfully utilized as a cell substrate for human vaccines. 17, 18 vero cells are still the first choice cell model for various types of life-threating emerging pathogens such as h5n1 influenza virus, 19 ebola haemorrhagic fever virus, 20 and middle east respiratory syndrome (mers) coronavirus. 21 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. 17 -19 in addition, antigenic drift affecting the vaccination efficacy to humans, which often occurs during the proliferation of influenza viruses in hen eggs, 22 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. 23 -25 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) 26 and multi-colour fluorescence in situ hybridization (m-fish) with 24 differentially labelled human chromosome-specific painting probes (24xcyte kit metasystems, altlussheim, germany). for detailed information, see supplementary data. genome dna was prepared from vero cells (with passage number 115) 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 hiseq2,000 (illumina inc., san diego, california). after quality filtering, sequences were assembled into scaffolds using sga and sspace software 27, 28 (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 29 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: rhemac2) and agm (chlorocebus sabaeus 1.0: gca_000409795.1) using the bwa-mem algorithm with default parameter settings. 30 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 54-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. 31 2.4. detection of genomic rearrangements in the vero jcrb0111 cell line copy number variants were detected using the control-freec software 32 with a 100-kb window size and 20-kb step size. sites with map quality scores ,40 were not used in the analysis. structural variants were identified using the integrated structural variant prediction method delly. junction sequences with !85% identity to the other part of the reference genome and split-read coverage .100 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 7 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 2, because one rearrangement had two non-rearranged regions. we selected the regions at which rearranged reads (split reads) consisted of at least 70% of total reads mapped on boundary regions (sum of canonical and split reads). we also filtered out the regions that had ,20 paired-end supports. for additional information, see supplementary data. genome landscape of vero cells [vol. 21, loss-of-heterozygosity (loh) regions were identified using 1-mb-size windows with average heterozygosity ,0.0005 and the ratio of homozygous to heterozygous snvs smaller than 0.2. the cut-off criteria were determined using the distribution of these values in a whole genome ( supplementary fig. s3 ). 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: dra002256). the full-length simian endogenous retrovirus sequences obtained in vero jcrb0111 cells have been deposited in ddbj (accession number: ab935214). 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 115 from the original primary culture started in march 1962) among the currently available stocks. lot jcrb0111 (hereafter referred to as vero jcrb0111) was expected to be a close relative to widely distributed vero cell seeds such as atcc ccl81 and who vero 10-87. 1,2,12,13,33 -35 the heteroplantation of vero jcrb0111 cells in immunocompromised nude mice (n ¼ 10) did not produce any discernible tumours, while that of human cervical carcinomaderived hela cells produced tumours in nude mice at 100% efficacy. thus, vero jcrb0111 cells are nontumourigenic, which is consistent with previous findings of vero cells being non-tumourigenic when their passage number was limited. 15, 16 when choosing the vero jcrb0111 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 jcrb0111 cell line had different karyotypes with chromosomes numbering between 52 and 62, and the modal chromosome number appeared to be 59 chromosomes in 79 of 100 metaphase cells (fig. 1a) . the same analysis on pbmc from normal female agm showed 60 chromosomes ( supplementary fig. s1a) . 36 because the g-banding karyotypes of vero cells include several abnormal chromosomes (fig. 1b) , m-fish was applied to identify chromosomal rearrangements. human m-fish probes hybridized efficiently to agm chromosomes and showed 32 syntenic blocks in normal agm karyotypes (fig. 1c ). this homology to humans was consistent with previous findings, 37 and implied that the human m-fish signal pattern can be used as a normal agm reference (fig. 1c ). g-banding and m-fish analyses revealed that 18 of 37 vero metaphases represented a main clone with 59 chromosomes. m-fish identified 40 segments in the vero metaphases ( fig. 1d) , which indicated the occurrence of one fusion and seven translocations. the fusion occurring between chromosomes 7 and 24 led to a reduction in the chromosome number from 60 to 59. duplications and deletions were also detected in 6 chromosomes (fig. 1d) , showing rearrangements involved in 14 chromosomes. however, major chromosomal rearrangements were not detected in the other 46 chromosomes by g-banding or m-fish ( fig. 1b 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 11 of 37 cells ( supplementary fig. s1c 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 (18 of 37 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 s1 . after quality filtering, we obtained a total of 2.55 billion paired-end and 390 million mate-pair reads. the filtered sequences were used for de novo assembly of the vero genome. our final scaffolds consisted of 401,905 scaffolds, which were 2.97 gb in total, and had the n50 of 508 kb and n90 of 48 kb. the augustus program identified 25,877 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. 1,2 however, the species classification of agms is a still debated issue and has been revised several times. 38, 39 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. 40 the mutation rate in the mitochondrial genomes of old world monkeys is known to be markedly higher than that in nuclear genomes 41 ; 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 s2 ). furthermore, a phylogenetic tree using whole mitochondrial genome (13) could not be distinguished using human m-fish probes, the copy number analysis revealed a gain at chromosome 15q (fig. 2b) , which indicated that der(13) had additional material over chromosome 15. genome landscape of vero cells [vol. 21, sequences indicated that the vero cell line was closest to c. sabaeus (fig. 2a) . 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. 1 ) and the almost diploid copy number of x chromosomes in sequence data (fig. 2b) were observed in vero jcrb0111 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 92% 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 !45 and coverage !10), which indicated that the rhesus macaque genome would work as a reasonable reference sequence for analysing the vero cell genome. approximately 58.5 million snvs were identified (supplementary table s3 ). of these, 51.2 million and 7.3 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 8-12 million years ago. 42, 43 the estimated divergence between the rhesus macaque and vero cell line genomes was 2.2%. the level of heterozygous snvs in the vero cell line was also high and was markedly higher than that in a human individual. 44 this may be partly explained by the higher genetic diversity within agm populations. we also identified 2.9 and 2.7 million small deletions ( 28 bp) and insertions ( 44 bp), respectively (supplementary table s3 ). vero cell line by comparing our paired-end reads with the publicly available draft genome of the agm c. sabaeus (c. sabaeus 1.0: gca_000409795.1), we examined potential .1kb-scale deletions, segmental duplications, inversions, and translocations using the information of improper paired-end mapping and split-read mapping. 45 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 138 deletions, 78 duplications, and 12 inversions of high frequency were detected ( fig. 2b; supplementary table s3 ), whereas none of the translocation candidates were detected at a high frequency. agm chromosome 12 harboured a homozygous deletion that spanned across nearly 9 mb (supplementary tables s3 and s4 ). the region was syntenic to human chromosome 9 and rhesus macaque chromosome 15 (from mllt3 to lingo2) and contained 40 genes including the type i interferon gene cluster and cdkn2 genes ( table s4 ). 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 2q, 8q, 15q, 16q, 21, 23q, 28, and xq had three copies, while eight regions at 3p, 7p, 13p, 17q, 21q, 27q, and xq were identified as a single copy (fig. 2b) . agm chromosome 8p showed an intermediate coverage between two and three copies, in which the duplication and translocation of agm chromosome 8p varied among the clones. these large-scale copy number changes agreed well with the karyotypes examined using m-fish analysis (fig. 1d) . 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 6q, 12q, and 23. the loh on chromosome 12 harboured a large homozygous deletion ( fig. 2c; supplementary table s4 ; see also below). the large deletions predicted by the massively parallel sequencing system were validated by genomic pcr. regarding the 8.85-mb deletion of chromosome 12, a set of pcr primers striding across the deletion junction produced a 230-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. 3a) . we tested two vero cell lines, jcrb0111 and atcc ccl81, and obtained identical results (fig. 3a) . in similar validation tests for another four large (.90 kb) predicted deletions (supplementary table s4 ), 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. 3a; supplementary fig. s2 ). four small (1-2 kb) predicted deletions were also confirmed to exist ( fig. 3b; supplementary fig. s2 ). analysis of collected srv-related short reads from all paired-end short reads of the vero jcrb0111 cell line, followed by analyses of gene assignment and long terminal repeat (ltr) finding, identified the 8,367 bp complete srv genome sequence. the medians of variant frequencies were 25.5 and 8.0% in the highly variable and env-deleted region (nucleotide position 7525 -7829) of srv, respectively (fig. 4) . the copy number of srv with 8.4-kb full length was estimated to be 30%, while that of srv with the deletion of the 7525 -7883 nucleotide (nt) region encompassing the c-terminal part in env and a portion of ltr was 70%. the srv-vero of jcrb0111 had 97% of the same nucleotides as those of atcc ccl81 (genbank_id: jn134185). the number of minor alleles was 703 for snvs and 1 for the insertion among the whole complete consensus srv sequence in the vero jcrb0111 genome. four minor mutation sites caused three nonsense mutations and one frameshift on the pol or env region (supplementary table s5 ). previous studies suggested a frameshift mutation in pol (position 3726) or frameshift mutation in prt (its position was not reported) on the srv proviral sequences of vero e6 or atcc ccl81 cells. 46, 47 however, our study did not detect equivalent mutations on the srv proviral sequences of vero jcrb0111 cells; however, other notable mutations were instead detected (supplementary table s5 ). 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, 48 which may be the main cause for the high susceptibility of these cells to various types of microbes. the homozygous deletion of aand b1-interferon genes in vero cells was previously reported using classical dna hybridization analysis. 49 the present study determined an 9-mb deleted region in chromosome 12 at the nucleotide level and further revealed an 59-mb loh around the deleted region ( fig. 2b and c) , which suggested that an 9-mb deletion first occurred in one of two homologous chromosome 12 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 12 of vero cells is syntenic to human chromosome 9p21 -p22, which contains many genes of type i interferons (a8, a2, a1/13, a6, a14, a4, a17, a21, v1, and b1) (supplementary table s4 ). the human syntenic region corresponding to the deleted region in agm chromosome 12 also contained cdkn2a and cdkn2b: cdkn2a encodes cyclin-dependent kinase (cdk) inhibitor 2a/p16 ink4a (which inhibits cdk6, a negative regulator of the retinoblastoma protein prb) and p14 arf (which inhibits the p53-negative regulator mdm2) in an alternate reading frame to the former, while cdkn2b encodes cdk inhibitor 2b/p15 ink4b (which inhibits another prb-negative regulator cdk4) (supplementary table s4 ). 50, 51 the cdk inhibitors and mdm2 inhibitor act as key regulators of the cell cycle, and mutations in cdkn2a-cdkn2b often occur in various types of human cancer; however, these mutations by themselves are not enough to transform 678 genome landscape of vero cells [vol. 21, cells into tumourigenic cells. 50 -52 the loss of both cdkn2a and cdkn2b 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. 15 although the karyological analysis demonstrated that vero cells had various chromosomal rearrangements (fig. 1c) , no translocation was identified in the wholegenome sequence (fig. 2b) . 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. 1c and d; supplementary fig. s1c and d) . haplotype sequencing may be necessary to determine such heterozygous events. many srv sequence variations existed in vero jcrb0111 cells (fig. 4) . as for srv associated with the vaccine-producing vero e6 cell line (the parental cell line of which is atcc ccl81), a frame-shifting single-nucleotide insertion in the polymerase gene was identified. 46 this frameshifting mutation was not detected in srv associated with the vero atcc ccl81 cell line 47 or vero jcrb0111 cell line (this study). srv variant sequences lacking the u3 and r regions of 3 0 ltr were instead detected in vero atcc ccl81-associated srv, 47 while the results of this study suggested that some srv copies in the vero jcrb0111 cell genome were defective in the env-3 0 ltr region (fig. 4) . 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 53 (the who guidelines on animal cell substrates are available at http://www.who.int/biologicals/vaccines/ trs_978_annex_3.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. 3) . metagenome analysis is a powerful approach that can be used to survey microbial contamination in biological pharmaceuticals. 46, 47, 54 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 jcrb0111 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. 21, 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 sv40 in tissue culture: preliminary step for cancer reserach in vitro studies on sv40 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 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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 1-interferon genes in human leukemia and derived cell lines the regulation of ink4/arf in cancer and aging epigenetic regulation of the ink4b-arf-ink4a 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-263439-oquk4t96 authors: park, jung-eun; cruz, deu john m.; shin, hyun-jin title: clathrinand serine proteases-dependent uptake of porcine epidemic diarrhea virus into vero cells date: 2014-10-13 journal: virus res doi: 10.1016/j.virusres.2014.07.022 sha: doc_id: 263439 cord_uid: oquk4t96 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, 2003; smith and helenius, 2004) . 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., 1981) . 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., 2002) . 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., 2001; pelkmans and helenius, 2002) . 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., 2006; simmons et al., 2005) . the porcine epidemic diarrhea virus (pedv) is classified as alphacoronavirus together with transmissible gastroenteritis virus (tgev), feline infectious peritonitis virus (fipv), and human coronavirus 229e (hcov-229e). pedv causes an acute watery diarrhea in suckling piglets, which results in approximately 50% mortality among suckling piglets and reduces the weight among fattening pigs (debouck and pensaert, 1980) . porcine epidemic diarrhea (ped) is first recognized in pigs in the united kingdoms in 1971 (wood, 1977) . although no evidence of ped is currently reported from canada, similar coronavirus-like particles were reported from herds in quebec in 1980 (turgeon et al., 1980) . 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, 2012) . recently, pedv is spreading rapidly in swine farms in the united states, resulting in high mortality in piglets in more than 17 states (mole, 2013 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., 2007; oh et al., 2003) . upon receptor binding, several coronaviruses in alphacoronavirus enter cells via endocytosis. for example, extensive studies on hcov-229e 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., 2004) . inside the endosome, cellular proteases that are active in a low-ph environment facilitate membrane fusion (kawase et al., 2009) . similarly, tgev binds to porcine apn (weingartl and derbyshire, 1994) , 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., 1998) . fipv also requires acidification of endosomes for successful entry (takano et al., 2008) . inhibition of fipv infection with nystatin, a pharmacological reagent that causes caveolae to flatten and disrupt the coat structure, and dynamin 2 inhibitor suggests that fipv entry might actually involve some types of caveolae-dependent endocytosis (van hamme et al., 2008) . 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 10% heat-inactivated fetal bovine serum (fbs, gibco), 100 u/ml penicillin, 100 g/ml streptomycin and 34 g/ml amphotericin b. kpedv-9, a vero cell-adapted korean strain, was propagated in vero cells as described previously (hofmann and wyler, 1988) . briefly, vero cells were inoculated with the kpedv-9 at a multiplicity of infection (moi) of 10 and cultured in serum-free mem for 72 h at 37 • c with 5% co 2 . the progeny viruses were titrated using the focus formation assay following a method described previously (cruz and shin, 2007) . kpedv-9 infection in vero cells under trypsin and non-trypsin conditions was compared for 48 h. vero cells in 4-well tissue culture (tc) plate (spl labware) were inoculated with kpedv-9 and cultured in either serum-free mem or mem supplemented with trypsin (10 g/ml). infection was stopped by addition of 5% paraformaldehyde (pfa) at the indicated times for immunocytochemistry. vero cells were treated with various concentrations of either cpz for 30 min or 0.45 m sucrose for 10 min to inhibit the formation of clathrin-coated pits. to block the caveolae-dependent pathway, cells were incubated with various concentrations of nystatin for 30 min. control cells were incubated with or without dimethyl sulfoxide (dmso). cells were inoculated with kpedv-9 at a moi of 10 for 2 h, and then overlaid with 0.5% methyl cellulose in mem containing trypsin for 10 h. at 10 hpi, pedv-infected cells were detected by immunocytochemistry. to prepare ultra-purified trypsin-free viruses, vero cells were inoculated with the kpedv-9 at a moi of 10 and cultured in serumfree mem for 72 h. supernatant was clarified by centrifugation at 20,000 × g for 20 min at 4 • c, followed by ultra-centrifugation using a 20% sucrose cushion at 150,000 × g for 3.5 h. following resuspension in buffer a (1 m tris, ph 8, 5 m nacl, 0.1 m cacl 2 ), protein concentration of purified virus stock was determined by the bradford assay. fluorochrome conjugation of kpedv-9 with alexa fluor 594 (af594) carboxylic acid-succinimidyl ester (molecular probes) was performed according to manufacturer's instructions. briefly, 5.0 mg of ultrapurified kpedv-9 was dialyzed in labeling buffer (0.1 m nahco 3 , ph 8.3) at 4 • c overnight. virus was then incubated for 1 h on a platform rocker at room temperature with 1 g of af594 succinimidyl ester in 100 l of dmso. the af594-labeled kpedv-9 was extensively dialyzed in buffer a. vero cells were prepared on cover glasses a day before assay. for af594-kpedv-9 co-localization with endocytic markers, the cells were incubated with af594-kpedv-9 combination with 10 g/ml of alexa fluor 488-conjugated transferrin (af488-tf) or 5.0 g/ml of alexa fluor 488-cholera toxin subunit b (af488-ct-b) for 30 min on ice to synchronize entry, and then shifted to 37 • c. unbound viruses were removed, and the cells were fixed in 2% pfa at indicated times and analyzed at magnification of 63× on the laser scanning confocal microscope. vero cells were treated with either 50 mm nh 4 cl or 1 g/ml baf-a1 to neutralize the intracellular ph. the cells were then inoculated with kpedv-9 at a moi of 10 for 2 h in the presence of lysosomotropic agents. the virus inoculums were removed by washing with pbs. cells were then overlaid with 0.5% methyl cellulose in mem containing trypsin for 10 h. at 10 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-9 and cultured in trypsin-free mem for 20 h. afterwards, the cell monolayer was washed thrice with pbs and replenished with serum-free mem adjusted to ph 3, 4, 5, 6, 7. mem containing trypsin (10 g/ml) at ph 7 was used as positive control. the cultures were further incubated at 37 • c for 4 h, and then fixed with 5% pfa. pedv-infected cells were detected by immunocytochemistry. cells were pretreated with various protease inhibitors such as e-64 (10 m), aebsf-hcl (500 m), pepstatin a (10 g/ml), and phosphoramidon (10 m) for 1 h. for examination the synergistic antiviral activity of aebsf-hcl and lysosomotropic agent, cells were treated with aebsf-hcl and/or nh 4 cl for 1 h. treated cells were then infected with kpedv-9 at a moi of 1 for 1 h in the presence of inhibitors. after 1 h adsorption, virus inoculums were removed by washing with pbs. cells were then overlaid with 0.5% methyl cellulose in mem containing trypsin for 10 h. at 10 hpi, pedv-infected cells were detected by immunocytochemistry. vero cells were pretreated with either 50 mm nh 4 cl or 10 g/ml cpz and then inoculated with kpedv-9 at a moi of 10 for 2 h. after adsorption, virus inoculums were removed by washing with pbs. cells were then overlaid with 0.5% methyl cellulose in mem containing trypsin for 10 h. at 10 hpi, pedv-infected cells were detected by immunocytochemistry. to detect expression of viral proteins, kpedv-9 infected vero cells were fixed with 5% pfa for 5 min and permeabilized with 1% np40. following three washes with pbs, cells were incubated with 1:5000 dilution of mouse anti-pedv polyclonal antibody for 1 h. cells were washed three times with pbs and then incubated with 1:1000 dilution of goat anti-mouse igg conjugated with horseradish peroxidase. kpedv-9 infected vero cells were stained using a 3, 3 -diaminobenzidine tetrahydrochloride solution containing nicl 2 and h 2 o 2 (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 6-well tc plates were treated with various chemicals to inhibit each endocytic pathway as described above. cells were inoculated with kpedv-9 at a moi of 10 in the presence of drugs. at 2 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 36 hpi. the extracted proteins were diluted in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) sample buffer and electrophoresed in 10% 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-1000plus. it has been reported that pedv requires an extracellular trypsin for its successful infection in vitro (kusanagi et al., 1992; lai and cavanagh, 1997; park et al., 2011) . 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, 1988) . 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., 2007) . 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. 1 , we found that pedv could infect vero cells even without trypsin treatment. initial infection was confirmed as early as 8 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 24 hpi, more than 95% of the cell monolayer had formed large multi-nucleated cells. in sharp contrast, pedv growth without exogenous trypsin did not involve syncytial spread. at 48 hpi, more than 99% 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, 1988; park et al., 2011) . 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., 2001; pelkmans and helenius, 2002) . 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-2 with the clathrin-coated pit lattice and thus inhibiting clathrindependent endocytosis or (ii) hypertonic 0.45% 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-9. to measure the inhibitory effect of virus entry, cells were overlaid with 0.5% methyl cellulose in mem containing trypsin for 10 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 10 hpi and normalized with "untreated cells" control. as shown in fig. 2a , pedv infection remarkably diminished (>90%) 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. 2b) , whereas ␤-actin expression was same. consistent with previous results, pedv infection with hypertonic sucrose treated was also significantly inhibited (fig. 2c ). 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. 3a) . likewise, the levels of pedv n protein synthesized virtually identical in both presence and absence of nystatin (fig. 3b) . 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, 2002) . vero cells were incubated with fluorescence-labeled pedv along with each marker, and then evaluated their subcellular localizations by confocal microscopy. at 4 h later, both endocytic markers, ct-b and tf, were located in the cytoplasm. co-localization was observed only between pedv and tf (fig. 4a ), but not with ct-b (fig. 4b) . to further confirm these findings, vero cells were treated with cpz and nystatin prior to pedv inoculation. as shown in fig. 4c , 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-9 and ct-b (green, b), was observed in the merged images. (c) colocalization of (yellow) kpedv-9 and tf was observed inside cells in the mock-and nystatin-treated vero cells. by contrast, cpz treatment inhibited internalization of both kpedv-9 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 4 cl, a relatively weak base accumulating inside endosomal vesicles, or baf-a1, 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 60% reduction at 10 hpi output titer by nh 4 cl (fig. 5a) . pedv replication was about 80% inhibited at concentrations as low as 100 nm of baf-a1. 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. 5b , low ph did not induce cell-cell fusion. syncytium formation was not observed in any cells under the condition between ph 3 and ph 6 (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. 6a , we found aebsf-hcl induced the strongest inhibitory activity with more than 90% inhibition. in contrast, e-64, pepstatin a, and phosphoramidon showed relatively lower inhibition with about 5-10% 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-2, 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., 2006; simmons et al., 2005) . to evaluate this for pedv s case, cells were treated with aebsf-hcl in combination with nh4cl prior to infection. as shown in previous results, pedv infection was significantly inhibited either by aebsf-hcl or nh 4 cl treatment (fig. 6b) . 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 (10 g/ml for 30 min) or nh 4 cl (50 mm for 2 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 4 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., 2006; qiu et al., 2006; simmons et al., 2005) . 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-2, and hcov-229e and mediated viral membrane fusion with endosomal membranes within endosomes (kawase et al., 2009; qiu et al., 2006; regan et al., 2008; simmons et al., 2005; turk et al., 1999) . similarly, various exogenous and cellular proteases such as trypsin, transmembrane proteases serine 2 enhance sars-cov entry by inducing virus-cell fusion at cell surface (glowacka et al., 2011; matsuyama et al., 2005 matsuyama et al., , 2010 shulla et al., 2011) . the block to infection mediated by lysosomotropic agents could be bypassed by treating with exogenous or cellular proteases (matsuyama et al., 2005; simmons et al., 2005) . pedv infection in vitro is also largely dependent on trypsin supplement (hofmann and wyler, 1988; park et al., 2011) . 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. 1) . 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. 2) . 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. 4) . 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 nh4cl or baf-a1, and then infected with kpedv-9. pedv entry was scored by immunocytochemistry at 10 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 4 h. low to neutral ph range (ph 3-7) 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 4 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, 2011) . 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., 2006; simmons et al., 2005) . similar to other coronaviruses, pedv infection was significantly inhibited by both nh 4 cl and baf-a1 (fig. 5a ), but acidic ph did not induce pedv s-mediated fusion (fig. 5b) , 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. 1) , 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. 6a ). 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. 6b , 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. 7 , 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-a59 s mediated infection study (wicht et al., 2014) . 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., 2004; follis et al., 2006) . 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 spike protein by a furin-like enzyme affects cell-cell but not virus-cell fusion experimental infection of pigs with a new porcine enteric coronavirus, cv 777 furin cleavage of the sars coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response the coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment propagation of the virus of porcine epidemic diarrhea in cell culture sars coronavirus, but not human coronavirus nl63, utilizes cathepsin l to infect ace2-expressing cells protease-mediated entry via the endosome of human coronavirus 229e isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate the molecular biology of coronaviruses porcine aminopeptidase n is a functional receptor for the pedv coronavirus infectious entry pathway of influenza virus in a canine kidney cell line efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss2 proteasemediated enhancement of severe acute respiratory syndrome coronavirus infection a distinct class of endosome mediates clathrin-independent endocytosis to the golgi complex human coronavirus 229e binds to cd13 in rafts and enters the cell through caveolae identification of a putative cellular receptor 150 kda polypeptide for porcine epidemic diarrhea virus in porcine enterocytes receptor-bound porcine epidemic diarrhea virus spike protein cleaved by trypsin induces membrane fusion endocytosis via caveolae insider information: what viruses tell us about endocytosis cell entry of enveloped viruses endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type 2 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. 20120008358, 2011(no. 20120008358, -0023942, 211-2006. we really appreciate for all the advices and technical supports from dr. fumihiro taguchi in nippon veterinary and life science university. key: cord-298922-k568hlf4 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 e6 cells infected with classic pedv strain cv777 by using quantitative proteomic technique date: 2015-06-15 journal: j virol methods doi: 10.1016/j.jviromet.2015.03.002 sha: doc_id: 298922 cord_uid: k568hlf4 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 e6 cells using mass spectrometry and an isobaric tag for relative and absolute quantification. a total of 126 proteins were identified that were differentially expressed between the pedv-infected and mock-infected groups (p < 0.05, quantitative ratio ≥1.2), among which the expression of 58 proteins was up-regulated and that of 68 proteins was down-regulated in the pedv-infected vero e6 cells, involving in integrin β2/β3, 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, rap1, autophagy, mitogen-activated protein kinase, pi3k-akt and jak-stat signaling pathways, and integrin β2/β3 and cystatin-c proteins, represented potential factors in pedv infection. our findings provide valuable insight into pedv-vero e6 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 90% in suckling piglets (pensaert and debouck, 1978) . ped was first reported in belgium and the united kingdom in 1978, and frequent outbreaks have occurred in various asian countries . since 2007, acute ped outbreaks have continually occurred in thailand, china, and the usa, which have resulted in substantial economic losses (puranaveja et al., 2009; li et al., 2012; chen et al., 2013; huang et al., 2013; marthaler et al., 2013; stevenson et al., 2013; yang et al., 2013; chen et al., 2014) . 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., 2012) . previous studies have investigated various control measures to protect against pedv infection, such as vaccines, diagnostic tools, and therapeutic drugs (sun et al., 2008; ren et al., 2011; sun et al., 2012; zhu et al., 2013; guo et al., 2013; kim and lee, 2013) . 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., 2014) . 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., 2008; boja et al., 2011; he et al., 2012; sun et al., 2013) . among current proteomics methods, quantitative high-throughput proteomics approaches are useful for the analysis of infection-associated proteins of pathogens (linde et al., 2013; papachristou et al., 2013; ye et al., 2013; zeng et al., 2015) . 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 e6 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 cv777 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 e6 cells, as previously described (hofmann and wyler, 1988) . the vero e6 cell-adapted pedv, the vero e6 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 e6 cells were cultured in dulbecco's modified eagle's medium containing 10% fetal bovine serum (fbs) in 75-cm flasks at 37 • c in a 5% co 2 atmosphere. when the cells reached 70-80% confluence, they were inoculated with the pedv at a multiplicity of infection of 1 in presence of 5 g/ml trypsin. at 48 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 7.4). a 1.5-ml aliquot of lysis buffer containing 4% sds, 1 mm dtt, and 150 mm tris-hcl (ph 8.0) was added to each flask, and the flasks were incubated at 37 • c for 5 min. the cell lysates were collected using a cell scraper, and boiled for 5 min. three cell lysate replicates were prepared for the pedv-infected (v1-v3) and mock-infected (c1-c3) vero e6 cells, and stored at −80 • c. western blotting was performed to confirm pedv infection by detecting the presence of the np of pedv in the vero e6 cells. aliquots of the cell lysates were subjected to sds-page on a 12% 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 5% (w/v) nonfat dried milk in pbs at 37 • c for 1 h, before incubation in pbs containing the anti-np monoclonal antibody (1:2000 dilution) at 37 • c for 1 h. after washing three times with 5% tween 20 in pbs (pbst), the membrane was incubated in pbst containing a horseradish peroxidase-conjugated goat anti-mouse igg (1:4000 dilution) at 37 • c for 1 h. after washing three times with pbs, the membrane was incubated with enhanced chemiluminescence detection reagents (biotopped, beijing, china) at room temperature for 3 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 (v1-v3) and mock-infected (c1-c3) vero e6 cells were pooled into the v and c samples, respectively, and western blotting was performed as described above, with the following exceptions: a 1:1000 dilution of the polyclonal antibodies anti-␤ tubulin, anti-integrin-␤3, anti-cystatin-c, anti-protein s100-a2, anti-apolipoprotein e4, and anti-centrin from rabbit (beijing biosynthesis biotechnology, beijing, china) was used as the primary antibody, and a 1:5000 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. (2009) . an aliquot of each cell lysate containing 200 g of protein was combined with 30 l of std buffer containing 4% sds, 100 mm dtt, and 150 mm tris-hcl (ph 8.0). the detergent, dtt, and other low-molecular-weight components were removed by dilution in ua buffer containing 8 m urea and 150 mm tris-hcl (ph 8.0) and repeated ultrafiltration using microcon (30 kda) ultrafiltration units. the reduction of cysteine residues was blocked by the addition of 100 l of 0.05 m iodoacetamide to the ua buffer. the samples were incubated for 20 min in darkness before ultrafiltration. the microcon filters were washed three times with 100 l of ua buffer, followed by two washes with 100 l ds buffer containing 50 mm triethyl ammonium bicarbonate (ph 8.5). the final protein suspensions were digested using 2 g of trypsin (promega, madison, wi, usa) in 40 l of ds buffer overnight at 37 • c, and the digested peptides were collected as the filtrate. the peptide content was quantified based on absorbance at 280 nm using an extinction coefficient of 1.1 for a 0.1 mg/ml solution. the digested peptide mixture was labeled using the 8-plex itraq reagent (life technologies, carlsbad, ca, usa), according to the manufacturer's instructions. each itraq reagent was dissolved in 70 l of ethanol, and added to the digested peptide mixture. the samples were labeled as c1-113, c2-114, c3-115, v1-116, v2-117, or v3-118 . 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 2 ml of buffer a containing 10 mm kh 2 po 4 in 25% acetonitrile (ph 2.7). the samples were loaded onto a 4.6 mm × 100 mm column packed with polysulfoethyl (5 m, 200å) chromatography resin (polylc, columbia, maryland, usa). the peptides were eluted at a flow rate of 1 ml/min using a gradient of 0-10% buffer b containing 500 mm kcl and 10 mm kh 2 po 4 in 25% acetonitrile (ph 2.7). the gradient elution consisted of 10-20% buffer b for 25 min, 20-45% buffer b for 5 min, and 50-100% buffer b for 5 min. the absorbance of the eluate was monitored at 214 nm, and fractions were collected at 1-min intervals. thirty fractions were combined into ten pools, and desalted using empore standard density spe c18 cartridges (sigma-aldrich, st. louis, mo, usa) with a bed diameter of 7 mm and a volume 3 ml. each fraction was concentrated by centrifugation in a vacuum, and reconstituted in 40 l of 0.1% (v/v) trifluoroacetic acid. all samples were stored at −80 • 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 (5 g) was loaded onto a c18-reversed phase column (15 cm × 75 m) packed with rp-c18 (5 m) resin in buffer a containing 0.1% formic acid, and eluted with a linear gradient of buffer b (80% acetonitrile and 0.1% formic acid) at a flow rate of 0.25 l/min for 140 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-10 method based on the selection of the most abundant precursor ions from the survey scan (300-1800 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 60 s. survey scans were acquired at a resolution of 70, 000 at m/z 200, and the resolution for the hcd spectra was set to 17, 500 at m/z 200. the normalized collision energy was 30 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 0.1%. the ms/ms spectra were compared to the uniprot cercopithecidae database (107 051 sequences, downloaded november 25, 2013) and a decoy database using the mascot search engine, version 2.2 (matrix science, london, uk), embedded in the proteome discoverer 1.4 software (thermo electron, san jose, ca). the following parameters were used for protein identification: a peptide mass tolerance of 20 ppm; an ms/ms tolerance of 0.1 da; trypsin digestion; a missed cleavage value of 2; the fixed modifications included carbamidomethyl, itraq8plex(k), and itraq8plex(n-term); the variable modification was oxidation; and an fdr value ≤0.01. protein quantification was performed using the proteome discoverer 1.4 software based on the centroided reporter ion peak intensity. the average quantitative value of each protein in samples c1, c2, and c3 (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., 2010) . 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 < 0.05), and the change ratio for the protein had to be ≥1.2 (yuan et al., 2012) . the expression of a protein with a v/c > 1.0 was considered to be up-regulated, and those with a v/c < 1.0 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 2007 software (microsoft, redmond, wa, usa). the deps were annotated using the blast2go, version 2.7.0, program (ashburner et al., 2000; quevillon et al., 2005; götz et al., 2008) . 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., 2012) . table 1 the proteins identified from pedv-infected and mock-infected groups. the vero e6 cells inoculated with pedv displayed distinct cpes at 48 h postinoculation, including cell shrinkage, cell fusion, and a rounded cell morphology, but no cells lysis or shedding was observed (fig. 1a) . the immunoblotting analysis confirmed that the vero e6 cells were pedv-infected. the band corresponding to the np of pedv was detected in samples v1, v2, and v3, whereas none was detected in samples c1, c2, and c3 (fig. 1b) . the identified peptides, identified proteins, quantified proteins, known/uncharacterized proteins, and the goc annotations are showed in table 1 . a total of 3178 proteins, including 15 564 peptides, were identified in the pedv-infected and mock-infected groups using the itraq-ms/ms approach, among which 3171 (99.78%) were quantified, 1859 (58.50%) were known proteins, and 1319 (41.50%) were uncharacterized/putative proteins. based on the gocs, 2061 (64.85%) of the proteins were annotated as biological process, 2495 (78.51%) were annotated as molecular function, and 1917 (60.32%) were annotated as cellular components. the quantification and significance of the identified proteins are shown in fig. 2 . the changes in the levels of expression between the two groups were analyzed based on statistical significance. of the 3178 proteins identified, 2496 (78.54%) were not differentially expressed (p > 0.05), and 675 (21.24%) were expressed at statistically different levels between the pedv-infected and mockinfected vero e6 cells (p < 0.05), including 357 proteins (11.23%) with a p-value between 0.01 and 0.05, 227 proteins (7.14%) with a p-value between 0.001 and 0.01, and 91 proteins (2.86%) with a p-value <0.001. the proteins with a p-value <0.05 were also filtered based on whether the v/c or c/v was ≥1.2. based on these criteria, a total of 126 (3.96%) of the 3178 identified proteins were determined to have been differentially expressed between the pedv-infected and mock-infected groups (table 2) . among the 126 deps, 46.03% (58/126) were up-regulated, and 53.97% (68/126) down-regulated. the known proteins and uncharacterized/putative proteins accounted for 69.05% (87/126) and 30.95% (39/126) of the deps, respectively. the dep displaying the greatest increase in expression in the pedv-infected vero e6 cells was isoform 2 of the ovarian cancer immunoreactive antigen domaincontaining 1 protein (1:2.5), and the dep displaying the greatest decrease in expression in the pedv-infected vero e6 cells was cystatin-c (1:2.2). the gene ontology (go) database has been widely used for describing protein function in a standardized format. according to their gocs, the 126 deps were annotated as cellular component, biological process, or molecular function. the go annotations are shown in table 2 , and distributions of the go annotations are shown in fig. 3 . seventy-eight deps were distributed among 16 groups of biological processes (fig. 3a) . the metabolic process (go:0008152), cellular process (go:0009987), single-organism process (go:0044699), and biological regulation (go:0065007) groups contained the highest proportions of the biological process deps. there were more up-regulated proteins in the cellular component organization group (go:0071840) than down-regulated proteins. seventy-four deps were distributed among eight cellular component groups (fig. 3b) , among which the organelle (go:0043226) and cell (go:0005623) groups contained the highest proportion of cellular component deps. there were more down-regulated deps in the membrane group (go:0016020) than up-regulated deps, and there were more up-regulated deps in the macromolecular complex group (go:0032991) than downregulated deps. ninety-seven deps were distributed among eight molecular function groups (fig. 3c) , among which the binding (go:0005488) and catalytic activity (go:0003824) 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 126 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. 4) . the annotations in the metabolism, organismal systems, and diseases pathway categories represented 32, 25, and 36 pathway groups, respectively (fig. 4a, 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. 4a ). the annotations in the organismal systems category included the tolllike receptor (tlr) signaling (ko04620), rig-i-like receptor (rlr) signaling (ko04622), and natural killer cell mediated cytotoxicity (ko04650) pathway groups (fig. 4b) , 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 (ko04142) pathway group, all ten of which were down-regulated deps (fig. 4c ). 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. 4d ). 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 (␤2 and ␤3 subunits) protein was annotated to the largest number of pathway groups (28), 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 e4 and centrin-2, two up-regulated deps integrin-␤3 and protein s100-a2, were selected to verify differential expression between the pedv-infected and mock-infected vero e6 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. 5) . in our study, pedv infection significantly alters protein expression in vero e6 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., 2010) . 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, rap1, pi3k-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. (2007) reported the rig-i signaling pathway plays an important role in antiviral innate immunity mechanisms in iecs. sheikh et al. (2013) reported the rap1a 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., 2013; wang et al., 2013) . our results showed that deps induced by pedv infection in vero e6 cells involved in the rlr, rap1, and jak-stat signaling pathways. it has been reported that the tlr, mapk, and pi3k-akt signaling pathways play roles in host cell responses to coronaviruses (mizutani et al., 2004; integrins are cell surface ␣/␤ heterodimeric glycoproteins that contribute to a variety of cellular functions (stewart and nemerow, 2007) . combinations of the various isotypes of the ␣ and ␤ subunits of integrins generate more than 20 different integrin proteins. previous studies have shown that various integrin molecules are used as receptors for virus attachment (stewart and nemerow, 2007; sun et al., 2013) . in our current study, the expression of integrin-␤2 and -␤3 was down-regulated and up-regulated, respectively, in response to pedv infection. the upregulation of integrin-␤3 expression is consistent with that observed in response to dengue virus infection (zhang et al., 2007) . our pathway analysis revealed that both integrin-␤2 and -␤3 are involved in 28 pathways that contribute to organismal systems, environmental information processing, cellular processes, and diseases. the integrin ␣v␤3 protein has been shown to serve as an entry receptor for various viruses (guerrero et al., 2000; neff et al., 2000; chu and ng, 2004; parry et al., 2005; wang et al., 2005) , some of which bind the integrin through an rgd sequence in a viral structural protein to initiate infection (stewart and nemerow, 2007) . 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 (1996) 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, 2013) . maier and britton (2012) 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 15 proteins, 12 (80%) 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 e6 cells, thus facilitating virus replication. previous studies have shown that the microtubule-associated protein 1b is a useful biomarker protein for autophagy (dong and levine, 2013) . we found that the expression of map1b was up-regulated 1.37-fold in the pedv-infected vero e6 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 oc43 and 229e (korant et al., 1986; collins and grubb, 1991) . 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., 1985) . shirato et al. (2011) reported the transmembrane type ii serine protease 2 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 e6 cells. apolipoprotein e4, galectin, clusterin, and transferrin receptor 1 have also been shown to be associated with virus infection (hishiki et al., 2010; peng et al., 2011; martin and uprichard, 2013; tripathi et al., 2013) , and may therefore function as infectionassociated proteins in pedv-infected vero e6 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 e6 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 e6 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 e6 cells are highly susceptible to pedv infection and facilitate experimental design and performance for proteomics, the vero e6 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 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pathways by virus infection cytotoxicity evaluation of oxidized single-walled carbon nanotubes and graphene oxide on human hepatoma hepg2 cells: an itraq-coupled 2d lc-ms/ms proteome analysis proteome analysis of porcine epidemic diarrhea virus (pedv)-infected vero cells up-regulated expression of beta3 integrin induced by dengue virus serotype 2 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. 31472209), the state national key laboratory of veterinary biotechnology (grant no. sklvbf201506/201302), and the program for new century excellent talents in heilongjiang provincial university (grant no. 1252-ncet-016). key: cord-256370-cz88t29n 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: 2016-02-29 journal: viruses doi: 10.3390/v8030065 sha: doc_id: 256370 cord_uid: cz88t29n 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 5' terminal sequence (5'-gguca) that is unique to all currently described viruses of the genus. the smallest genome segment is bicistronic encoding for a 14 kda protein similar to p14 membrane fusion protein of bush viper reovirus and an 18 kda protein similar to p16 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 [1, 2] , coronaviruses [3, 4] , paramyxoviruses [5, 6] and lyssaviruses [7, 8] . other viruses, without a known human disease link, have also been detected recently [9] [10] [11] . 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 [12] . bats are parasitized by a number of ectoparasites, including mites, bat flies, ticks and fleas, and often by some or all of these simultaneously [13] . 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 [13] [14] [15] . both bat fly families are hematophagous and potentially capable of (qiagen). cellular debris was removed by centrifugation at 14,000ˆg for 3 min, and the supernatant used for subsequent virus isolation and nucleic acid extraction procedures. viruses 2016, 8, x 5mm stainless steel beads (qiagen). cellular debris was removed by centrifugation at 14,000 × g for 3 min, and the supernatant used for subsequent virus isolation and nucleic acid extraction procedures. the wells of 24-well tissue culture plates (nunc) were seeded with vero e6 cells and grown to 80%-90% confluency in eagle's minimum essential medium (emem, lonza) supplemented with antibiotics (penicillin/streptomycin/amphotericinb, lonza) and 10% foetal calf serum at 37 °c and 5% co2. culture medium was removed and the monolayers in individual wells inoculated with 200 μl of ectoparasite pool homogenates (one pool representing parasites from one bat). after one hour adsorption at 37 °c, the inoculum was removed and fresh emem containing antibiotics and 2% foetal calf serum added. the 24-well plates were incubated for 14 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 200 μl of undiluted supernatant from the preceding passage. supernatants were collected from all wells displaying cpe after three blind passages, a 1/10 dilution prepared in emem, and 1 ml of this used to inoculate a 25 cm 2 tissue culture flask. if the same cpe was noted in the sub-cultured 25 cm 2 flask, a 1/100 dilution of this supernatant was prepared and used to inoculate a 75 cm 2 tissue culture flask for preparation of stock virus. stock virus titres were determined by standard tissue culture infectious dose 50 (tcid50) titrations on 96-well microtitre plates as described previously [36] . the wells of 24-well tissue culture plates (nunc) were seeded with vero e6 cells and grown to 80%-90% confluency in eagle's minimum essential medium (emem, lonza) supplemented with antibiotics (penicillin/streptomycin/amphotericinb, lonza) and 10% foetal calf serum at 37˝c and 5% co 2 . culture medium was removed and the monolayers in individual wells inoculated with 200 µl of ectoparasite pool homogenates (one pool representing parasites from one bat). after one hour adsorption at 37˝c, the inoculum was removed and fresh emem containing antibiotics and 2% foetal calf serum added. the 24-well plates were incubated for 14 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 200 µl of undiluted supernatant from the preceding passage. supernatants were collected from all wells displaying cpe after three blind passages, a 1/10 dilution prepared in emem, and 1 ml of this used to inoculate a 25 cm 2 tissue culture flask. if the same cpe was noted in the sub-cultured 25 cm 2 flask, a 1/100 dilution of this supernatant was prepared and used to inoculate a 75 cm 2 tissue culture flask for preparation of stock virus. stock virus titres were determined by standard tissue culture infectious dose 50 (tcid 50 ) titrations on 96-well microtitre plates as described previously [36] . for electron microscopy specimen preparation, 80%-90% confluent vero e6 monolayers in 25 cm 2 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 (3000ˆg for 5 min), and subsequently fixed in an equal volume of 2.5% glutaraldehyde in 0.1 m hepes buffer (ph 6.9) for visualization of virus particles by negative staining. a beckman airfuge ® (beckman coulter, brea, ca, usa) was used to concentrate all samples (10 min at 207 kpa), after which droplets of sample were adsorbed to 0.25% formar-coated copper grids for a minimum of 10 min, rinsed twice in deionised, distilled water and stained briefly in 2% phosphotungstic acid (ph 6.9). for ultramicrotomy, the remaining infected monolayers were flooded with the same fixative overnight, then routinely processed (postfixation in 1% buffered osmium tetroxide, graded ethanol dehydration, infiltration with a low viscosity resin (agar scientific, stansted, uk) and overnight polymerisation at 70˝c). seventy nm sections were cut on a leica em-uc6, double stained with saturated uranyl acetate and lead citrate, and viewed at 80 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 100 µl supernatant to 300 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 [37] . rnas were converted to cdna and amplified using sispa as described previously with modifications [38] . to enhance coverage of the terminal ends, an oligo containing three rgtp at the 3' 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 5' 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 500 (illumina) using either a 2ˆ150 or 2ˆ250 version2 kit. illumina and sispa adapter sequences were trimmed from the sequencing reads using cutadapt-1.2.1 [39] , quality filtering was conducted with prinseq-lite (-min len 50-derep 14-lc method dust-lc threshold 3-trim ns left 1-trim ns right 1-trim qual right 15) [40] and reads were assembled into contigs using ray meta with kmer length = 25 [41] . resultant contigs were aligned to the ncbi sequence database using blast. the mega (version 6) program was used to prepare alignments (clustalw) of nucleic acid segment sequences, deduced amino acid sequences, phylogenetic trees and pairwise distance calculations [42] . 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 1 . the evolutionary history was inferred by using the maximum likelihood method based on the jtt matrix-based model [43] . 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 (1000 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 mega6 [42] . 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.: lco1490 and hco2198 [13] . polymerase chain reaction was carried out in 50 µl reactions containing 25 µl mytaq red mix 2ˆ(bioline, london, uk), 2 µl of forward and reverse primer (10 µm), dna template (10 µl) and nuclease free water (11 µl). amplification steps were 94˝c for 5 min, 25 cycles of 94˝c for 60 s, 48˝c for 60 s and 72˝c for 90 s, and 72˝c for 10 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 e6 (source african green monkey kidney) and c6-36 (source aedes albopictus mosquitoes). cells were grown to 50%-70% confluency in 25 cm 2 flasks, supernatant removed and respective flasks inoculated with 1 ml of 10´1, 10´2, 10´3 and 10´5 dilutions from stock virus (1ˆ10 6 tcid 50 /ml) in emem. after 1 h adsorption at 37˝c (veroe6) or 28˝c (c6-36), the inoculum was removed, cells washed with 5 ml phosphate buffered saline (pbs) and fresh emem, antibiotics and 2% foetal calf serum (hyclone, logan, ut, usa) added. cultures were incubated for 13 days at 37˝c (veroe6) or 28˝c (c6-36) while 0.5 ml aliquots of supernatant were collected from each flask directly after inoculation and addition of fresh medium (day 0), followed by day 4, 7 and 13. rna was extracted from 140 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_796f (5'-tagtggttcgtatgcgtggt-3'), reverse morv_893r (5'-aacagccattcaatctcagg-3') and probe morv_875p (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 (50˝c for 30 min), hot-start taq activation (95˝c for 15 min) and 50 cycles of amplification (95˝c for 15 s; 52˝c for 25 s plus signal acquisition; 72˝c for 20 s). rna extracted from diluted stock mahlv (final 1ˆ10 5 tcid 50 /ml) was used as a qualitative positive control in each run. from a total of 273 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 2 ). the parasite pool that yielded mahlv isolate 2511 was collected from an apparently healthy adult female rousettus aegyptiacus bat captured at mahune cave in may 2013. cpe in vero cells were noted after two blind passages, and the supernatant collected on day five from passage four in a 75 cm 2 flask containing infected vero cells yielded 1ˆ10 6.25 tcid 50 /ml of the unknown virus. the second parasite pool that yielded mahlv isolate 06-24 was collected from an apparently healthy juvenile male rousettus aegyptiacus bat captured at mahune cave in june 2013. cpe in vero cells were noted after three blind passages, and the supernatant collected on day five from passage five in a 75 cm 2 flask containing infected vero cells yielded 1ˆ10 6 tcid 50 /ml of the virus. these supernatants, passage four of 2511 and passage five of 06-24, 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 3 ) [44] . sequencing of the cytochrome c oxidase subunit i gene (coi) and alignment to sequences available on genbank, followed by phylogenetic analysis (figure 4 ) 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 5 ). two-layered capsids with an outer diameter of 70-75 nm (n = 30) and an inner core of 42-45 nm, possessed clearly defined solvent channels radiating outwards through the clustered capsomers of the outer capsid layer ( figure 5 ). 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 81 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 5 ). two-layered capsids with an outer diameter of 70-75 nm (n = 30) and an inner core of 42-45 nm, possessed clearly defined solvent channels radiating outwards through the clustered capsomers of the outer capsid layer ( figure 5 ). 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 81 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 e6 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 e6 cells were multinucleate cells with extensive nuclear lobing, and cytoplasmic inclusion bodies associated with developing, double-shelled virus particles ( figure 6 ). 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 e6 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 e6 cells were multinucleate cells with extensive nuclear lobing, and cytoplasmic inclusion bodies associated with developing, double-shelled virus particles ( figure 6 ). tem observations therefore suggested that the isolated virus belonged to the reoviridae, sub-family spinareovirinae. evident in ultrathin sections of infected vero e6 cells were multinucleate cells with extensive nuclear lobing, and cytoplasmic inclusion bodies associated with developing, double-shelled virus particles ( figure 6 ). 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 10 segments. a polyetheleneglycol (peg) precipitated preparation of isolate 06-24 yielded the most viral specific reads and formed 11 contigs aligning to othoreoviruses using blastn and blastx. both the 5' and 3' 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 500. 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 7) 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 8-10) . 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-63.7%; rdrp amino acid identity-66.3%) while the closest bat-associated virus is broome virus (lambdab nucleic acid identity-60.7%; rdrp amino acid identity-58.0%) ( table 2) . homology of the divergent major outer capsid protein of mahlv to known orthoreoviruses is much lower: sigma b nucleic acid identity-28.7%-41.9%; amino acid identity-5.6%-24.3% (table 3) . the genome segments of mahlv were named according to the nucleotide length, which is consistent with the nomenclature of other orthoreoviruses [26] . a summary of the mahlv genome is given in table 4 . the total genome size is 23,200 nucleotides and predicted to encode eleven proteins, seven of which are structural. all ten genome segments of mahlv contain an identical 3' terminal sequence, ucauc-3', which is conserved between all known species of orthoreovirus, and an identical 5' terminal sequence, 5'-gguca which is unique to mahlv. non-coding regions (ncrs) are present at both ends of the genome segments, with the 5' ncrs being shorter in nucleotide length than 3' ncrs. the nucleotide sequences of the two isolates of mahlv, 2511 and 06-24, are not identical. nucleotide homology of the rdrp encoding segment between the two isolates is 93.5% (99.8% deduced amino acid sequence), and 80.4% (89.0% 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 l2, l3, m1, m3, s1, s2 and s3 each contain a single start aug codon in close proximity to the 5' end. the l1 segment of mahlv contains two aug start codons in close proximity to the 5' end, at positions 14 and 19. agcaugg) . the open reading frame initiating at position 29 is 2031 nucleotides in length and putatively encodes for an outer capsid protein involved in membrane penetration during infection (mub). the second aug initiates a 492 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 s1) contains the fusogenic orthoreovirus-wide conserved arginine amino acid at position 273. the s4 segment is bicistronic and encodes for a 14 kda protein similar to p14 membrane fusion protein of bush viper reovirus and a non-overlapping 18 kda protein similar to p16 non-structural protein of baboon orthoreovirus without a known function ( table 4 ). the isoelectric point of mahlv p18 is acidic (5.02), similar to that of p16 of broome virus and baboon orthoreovirus and contrary to that of other orthoreoviruses. the first ten amino acids in the putative 14 kda protein of mahlv are identical to the first ten amino acids in the p13 fusion protein of broome virus and represent the myristoylation consensus sequence required for fusion activity of the protein. the s4 segment does not encode a cell attachment protein, an observation also characteristic of broome virus and baboon orthoreovirus. 1 63.0 54.3 53.9 63.1 63.8 63.3 63.6 64.7 mahlv replicated efficiently in vero cell culture, with the inoculum containing a high dose of virus (10 5 tcid 50 /ml) leading to rapid monolayer destruction after inoculation, with a peak in virus rna (measured by real-time rt-pcr) by day 7, followed by a decrease on day 13. the inoculums containing a lower virus dose (10 4 and 10 3 tcid 50 /ml) resulted in a peak of rna detection on day 13. all three above-mentioned inoculum doses yielded detectable virus rna by day 4 after inoculation. the inoculum containing 10 1 tcid 50 /ml virus did not generate detectable viral rna until day 7, and was still showing an upward trend on day 13 (last sampling day). the virus did not replicate in the insect cells (c6-36) up to day 13, 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 [13] . 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 [19] . for conserved core proteins, an amino acid identity >85% for homologous proteins indicates that two viruses belong to the same species, while identity <65% indicates a possible new species. when comparing the amino acid sequence of more divergent outer capsid proteins, >55% identity indicates one species and <35% indicates different species. nucleic acid sequence identity of homologous segments of >75% indicates the same species and <60% a new species. the nature of conserved genome segment termini sequences of orthoreoviruses is also useful for virus classification [45] . the divergence of mahlv sequence from other known orthoreoviruses combined with a unique conserved 5' 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 s4 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 c6-36 cells in this study but aedes albopictus, from which the c6-36 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 [46] , and the strict host preference and specificity of bat flies [13] [14] [15] , 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 e6 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 [22, 23, 32, 33] . 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 cell line. in conclusion, we have identified a novel orthoreovirus which we propose should constitute a new species within the genus. two virus strains were isolated from ectoparasitic bat flies collected from egyptian fruit bats from a south african cave roost. this represents the first isolation of an orthoreovirus from arthropods and the first african virus in this genus with an indirect link to bats. fruit bats as reservoirs of ebola virus marburg virus infection detected in a common african bat bats are natural reservoirs of sars-like coronaviruses middle east respiratory syndrome coronavirus in bats, saudi arabia nipah virus: a recently emergent deadly paramyxovirus isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus isolation of rabies virus from an insectivorous bat (tadarida mexicana) in california fatal case of human rabies (duvenhage virus) from a bat in kenya: the netherlands detection of a novel herpesvirus from bats in the philippines a novel rhabdovirus isolated from the straw-colored fruit bat eidolon helvum, with signs of antibodies in swine and humans isolation and molecular characterization of fikirini rhabdovirus, a novel virus from a kenyan bat bats: important reservoir hosts of emerging viruses evolutionary history of indian ocean nycteribiid bat flies mirroring the ecology of their hosts bat flies (diptera: streblidae, nycteribiidae) parasitic on bats (mammalia: chiroptera) at parque da cantareira, sao paulo, brazil: parasitism rates and host-parasite associations bat flies-obligate ectoparasites of bats bartonella spp. in fruit bats and blood-feeding ectoparasites in madagascar global distribution and genetic diversity of bartonella in bat flies (hippoboscoidea, streblidae, nycteribiidae) detection of rhabdovirus viral rna in oropharyngeal swabs and ectoparasites of spanish bats virus taxonomy. classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses a new member of the pteropine orthoreovirus species isolated from fruit bats imported to italy structure and cytopathic effects of nelson bay virus a previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans identification and characterization of a new orthoreovirus from patients with acute respiratory infections isolation and characterization of three mammalian orthoreoviruses from european bats isolation and identification of a natural reassortant mammalian orthoreovirus from least horseshoe bat in china broome virus, a new fusogenic orthoreovirus species isolated from an australian fruit bat isolation and genomic characterization of a novel orthoreovirus from a brown-eared bulbul (hypsipetes amaurotis) in japan discovery of an orthoreovirus in the aborted fetus of a stellar sea lion (eumetopias jubatus) high similarity of novel orthoreovirus detected in a child hospitalized with acute gastroenteritis to mammalian orthoreoviruses found in bats in 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 6.0 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 10 genes of reovirus serotype 3 (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 2016, 8, 65 key: cord-270683-982eqtog 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 2 in turkey date: 2020-09-16 journal: plos one doi: 10.1371/journal.pone.0238614 sha: doc_id: 270683 cord_uid: 982eqtog coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and associated with severe respiratory illness emerged in wuhan, china, in late 2019. 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-19. we isolated sars-cov-2 from the nasopharyngeal sample of a patient in turkey with confirmed covid-19. we determined that the vero e6 and ma-104 cell lines are suitable for supporting sars-cov-2 that supports viral replication, development of cytopathic effect (cpe) and subsequent cell death. phylogenetic analyses of the whole genome sequences showed that the hcov-19/turkey/eragem-001/2020 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 [1] . transcription of coronaviruses requires a polymerase template switch, characterized as a discontinuous process unique among rna viruses [2] [3] [4] . based on the difference in protein sequences, covs are classified into four genera, alpha-cov, beta-cov, gamma-cov, and delta-cov2 [1, 2, 5] . there are hundreds of coronaviruses are circulating broadly among mammals and birds that cause respiratory, enteric, hepatic, and neurologic diseases [1, 6, 7] . until recently, six coronavirus species have been known to cause disease in humans. the 229e, oc43, nl63 and hku1 viruses are prevalent and cause mild illness, such as the common cold [1, 8] . 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 2002 and disappeared by 2004, and mers (middle east respiratory syndrome), which emerged in 2012 and continues to circulate in the middle east [9] [10] [11] [12] [13] . at the end of 2019, severe pneumonia cases of unknown etiology were reported in wuhan, a city in the hubei province of china [14] [15] . sequencing analysis revealed that this unidentified pneumonia was considered to be caused by a novel coronavirus [14, 16] . the world health organization (who) termed the disease as coronavirus disease-2019 (covid-19) on february 11, 2020 [17] . on the same day, the international committee on taxonomy of viruses (ictv) named this novel coronavirus as severe acute respiratory syndrome coronavirus 2 (sars-cov-2). sars-cov-2 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-2 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-19 outbreak has quickly expanded to worldwide [18] [19] [20] . it was declared a pandemic by the who on march 11, 2020. as of august 18, 2020, a total of 21,756,357 confirmed cases of covid-19 and 771,635 deaths have been reported in more than 200 countries and territories. (https://www.who.int/emergencies/diseases/novelcoronavirus-2019/situation-reports/). the first case of covid-19 in turkey was confirmed on march 112020. as of august 18, 2020, there have been 251,805 cases and 6,016 deaths (the ministry of health, turkey). in this study, we report the isolation of the hcov-19/turkey/eragem-001/2020 strain from a patient in turkey with confirmed covid-19. the whole genomic sequence and replication characteristics of the hcov-19/turkey/eragem-001/2020 strain are described. this is the first known report of the isolation and characterization of sars-cov-2 from a human clinical sample in turkey. the successful isolation and characterization of the virus will be essential for continued investigations of sars-cov-2 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 (2020-3-/23), 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 e6, atcc crl-1586), rhesus monkey kidney cells (ma-104, atcc crl-2378), human adrenal carcinoma cells (sw-13, atcc ccl-105) and human cervical adenocarcinoma cells (hela, atcc ccl-2) were cultured in dulbecco's modified eagle's medium (dmem) (sigma, germany) supplemented with 10% heat-inactivated fetal bovine serum (fbs) (gibco, usa), 100 mm l-glutamine, 100 u/ml penicillin, 100 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 17, 2020 due to respiratory symptoms. the patient's nasopharyngeal sample was obtained by using a utm™ kit containing 1 ml of viral transport media (copan diagnostics, usa) on day 4 of his illness. the diluted sample was inoculated onto monolayers of vero e6 cells and gently agitated at 37˚c for 1 h. consequently, dmem with 2% 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 3 enhanced facility (bsl-3). viral rna was isolated from 140 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 60 min at 42˚c, and the reaction was stopped by heating the mixture at 95˚c for 5 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) [21] . the pcr was conducted in a 50 μl reaction mixture containing 3 μl of cdna template, 10 mm tris-hcl, 50 mm kcl, 1.5 mm mgcl 2 , 2019-ncov_n1 forward primer (5 0 -gaccccaaaatcagcgaaat-3 0 ), 2019-ncov_n3 reverse primer (5 0 -tgtagcacg att gcagcattg -3 0 ), 1 u of taq polymerase (thermo scientific, usa), and 1.25 mm dntps. the cycling conditions were 94˚c for 3 min, followed by 35 cycles of 94˚c for 45 sec, 55˚c for 45 sec, and 72˚c for 1 min with a final extension at 72˚c for 10 min. the pcr products were visualized by ethidium bromide staining after 1% 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 2019-ncov_n1 forward primer (5 0 -gaccccaaaatcagc gaaat-3 0 ), 2019-ncov_n2 reverse primer (5'-gcgcgacattccgaagaa-3') and 2019-ncov_n3 forward primer (5'-gggagccttgaatacaccaaaa-3'), and 2019-ncov n2 reverse primer (5'-gcg cgacattccgaagaa-3'), respectively. twenty-four-well plates were seeded with vero e6 cells and incubated at 37˚c with 5% co 2 . the monolayer was inoculated with 10-fold serially dilutions of the virus. after incubation for 1 h at 37˚c with shaking, the monolayer was overlaid with 0.5 ml overlay medium containing 0.3% low melting agarose (sigma, germany). after incubation at 37˚c for 3 days, the cells were fixed with 10% formalin (v/v) for 90 min at room temperature. the agarose overlay was discarded, and the plaques were visualized by staining the monolayer with 1% crystal violet (w/v) in 20% ethanol (v/v). we cultured to sars-cov-2 passage 1 (1:100 dilution) in vero e6 cells to make virus the passage 2 virus stock. the sars-cov-2 virus lysate was then harvested at 48 h post-infection and the supernatants were collected, clarified, and stored at -80˚c. to determine the titer of the passage 2 virus a focus forming assay (ffa) was performed as described previously [22] . briefly, vero e6 cells were seeded on 96 well-plates and incubated at 37˚c with 5% co 2 for overnight. the cell monolayers were inoculated with 10-fold serial dilutions virus at 2nd passage. the diluted samples were added in triplicate to confluent vero cell monolayers. after absorption for 1 h at 37˚c, the supernatants were removed and the cells were washed with pbs. the cell monolayers were overlaid with virus medium containing 1% cmc (carboxymethyl cellulose) then incubated at 37˚c with 5% co 2 for 72 h. after fixation with 10% neutral buffered formaldehyde at room temperature for 20 min, the cells were permeabilized with 0.1% triton x-100 in pbs for 20 min with gentle rocking and blocked with 5% skim milk in pbs. the wells were then incubated with a human antibody to sars-cov-2 nucleocapsid protein (1:2500) (genscript; hc2003) for 1h in tbst (100 mm tris-hcl ph 8.0, 1.5 m nacl, 1% tween 20) at 37˚c and washed 3 times with tbst. the cells were incubated for 1 h with goat anti-human igg conjugated to fluorescein isothiocyanate (fitch) (southern biotech, usa) and diluted 1:1000 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 [22] . to obtain the virus passage 3 virus stock, the vero e6 cells were infected with the virus passage 2 virus at an moi of 0.01, and the viral lysate were was harvested at 48 h post-infection and the supernatants were collected. subsequently, the virus passage 4 virus was generated in vero e-6 cells infected with virus passage 3 virus at an moi of 0.01. cell lysates were harvested in laemmli sodium dodecyl sulfate-polyacrylamide (sds) gel electrophoresis sample buffer containing 2% sds and 5% β-mercaptoethanol. the lysates were boiled and loaded onto a polyacrylamide gel. the samples were separated on 12% resolving and 5% stacking sds-page gels in a mini electrophoresis unit (bio-rad, usa) at 100 v for 1 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 5% skimmed milk, the membrane was incubated either with a rabbit polyclonal to sars-cov-2 spike glycoprotein (1/1000) (abcam; ab272504) or a human antibody to the sars-cov-2 nucleocapsid protein (1:2500) (genscript; hc2003) followed by a goat anti-rabbit horseradish peroxidase (hrp)-conjugated antibody (1:2000 dilution, invitrogen; usa) and a goat anti-human horseradish peroxidase (hrp)-conjugated antibody (1:2000 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 1000a, sigma germany). vero e-6 and ma-104 cells cultured in 24-well-plates were infected with an moi at an 0.1 (passage 4 virus). the cultures were harvested by scraping cell monolayers from at different time points (6, 12, 18, 24, 48 and 72 h) and stored at -80˚c. the vero e-6 cells were then inoculated with 10-fold serial dilutions of the samples in triplicate per dilution. the viral inoculum was removed. the cell monolayers were overlaid with virus medium containing 1% cmc. the cells were fixed with 10% neutral buffered formaldehyde after infection of 72 h at room temperature for 20 min, and permeabilized with triton x-100. the wells were then incubated with a human antibodt to the sars-cov-2 nucleocapsid protein (1:2500) (genscript; hc2003) for 1h in tbst at 37˚c and washed 3 times with tbst. the cells were incubated for 1 h with goat anti-human igg conjugated to fluorescein isothiocyanate (fitch) (southern biotech, usa) and diluted 1:1000 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-19/turkey/eragem-001/2020, vero e6 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 26 dna amplicons the from full genome amplification [23] 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 500 (illumina, usa) platform with a 2x150 cycle kit (gen era diagnostics inc., turkey). the quality of the raw data was evaluated by fastqc v.0.11.5 (babraham bioinformatics) and low-quality bases, primers and remnant adapters were trimmed using trimmomatic v.0.32 [24] . the reads were aligned to the previously assembled sequence of the sars-cov-2 genome (genbank accession: mn908947.3) using the burrows-wheeler aligner v.0.7.1 with the mem algorithm [25] . the variants were called by using genome analysis toolkit (gatk) v.3.8.0 with the haplotypecaller algorithm [26] and were manually inspected in genomebrowse v2.1.2 (goldenhelix). the variants that had low quality and a low variant fraction (%<60) were filtered. the filtered variants and reference sars-cov-2 genome were used to generate the consensus sequence using bcftools v1.9 [27] . for multiple sequence alignment, complete (>29,000bp) and high-coverage genomes (n = 3970) were used from the gisaid database. the gisaid strain genomes including the genome of our strain were aligned using the mafft v7.450 tool [28] . phylogenetic analysis of the alignment was performed using the iq-tree v. 1.6.12 with a general time-reversible (gtr) model [29] . the whole genome sequence was submitted to genbank (id:mt327745.1) and gisaid (id: epi_isl_424366) and the raw data deposited on sra (samn15062833). graphpad prism 7 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 0.05 where � p<0.05. after 24 h of incubation, very little but visible cpe was detected in virus passage 1 virus ( fig 1b) . the time for the onset of cpe was typically 48 h post-infection ( fig 1c) and major cpe was observed within 72 h post-infection ( fig 1d) . 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 1a, 1b, 1c and 1d) . as we observed cpe in the infected monolayers, rt-pcr was used as a confirmatory assay. the primer set from cdc [21] targeting the nucleocapsid protein gene (np) of sars-cov-2 was used for the pcr reactions. a pcr product size of 469 bp was amplified with the 2019-ncov_n1 forward and 2019-ncov_n3 reverse primers (fig 1e, lane 2) . we amplified two pcr products, with sizes of 945 bp and 549 bp, with the 2019-ncov_n1 forward, and 2019-ncov_n2 reverse primers and with the 2019-ncov_n3 forward and 2019-ncov_n2 reverse primers, as in shown fig 1e lane 3 and fig 1e lane 4 , respectively. we also performed to plaque assay to purify of sars-cov-2 for subsequent use in further experiments. representative sars-cov-2 plaques in the vero e6 cell monolayers infected with sars-cov-2 are shown in fig 1f and 1g . taken together, these results suggest that a sars-cov-2 strain named hcov-19/turkey/eragem-001/2020 was successfully isolated from the nasopharyngeal sample of a patient in turkey with confirmed covid-19. we cultured to sars-cov-2 passage 1 in vero e6 cells to make the virus passage 2 virus stock. subsequently, the passage 2 virus stock was passaged two more times in vero e6 cells. the virus stocks were quantified by using ffa (fig 2) . we determined that the titer of the passage 2 virus was 2.8x10 4 ffu/ml, while the titers of the passage 3 and passage 4 viruses were 4.3x10 5 ffu/ml and 4.9x10 6 ffu/ml, respectively (fig 2) . these results indicated that propagation of the hcov-19/turkey/eragem-001/2020 strain in vero e6 cells led to an increasing in the viral titers in each passage. in addition to vero e6 cells, we examined the susceptibility of ma-104, sw-13 and hela cell lines to infection by sars-cov-2. all cell lines were infected with an moi of 0.1 (virus passage 4 virus) and monitored for cpe until 72 h post-infection. only the ma-104 cell line developed cpe. in abnormal areas, small clusters of rounded cells, cell detachment and degeneration were observed (fig 3) . similar to vero e6 cells infected with sars-cov-2, cpe formation in the ma-infected cells began at 24 h post-infection ( fig 3b) and increased at 48 h post-infection (fig 3c) . the complete cpe was observed within 72 h post-infection (fig 3d) . to confirm the results of the susceptibility of the ma-104, sw-13 and hela cell lines to infection by sars-cov-2, all cell lines were infected with an moi of 0.1 (passage 4 virus) and incubated at 48 h post-infection. an immunofluorescence assay (ifa) confirmed that the vero isolation of sars-cov-2 in turkey e6 and ma-104 cell lines supported the replication of sars-cov-2. in contrast, sars-cov-2 did not replicate in sw-13 and hela cells. (fig 4) . to expand these observations, we examined the expression of the sars-cov-2 proteins. all cell lines were infected with an moi of 0.5 (passage 4 virus). cell lysates from infected cell lines were harvested at 24 h post-infection and were probed either with the rabbit polyclonal antibody to sars-cov-2 spike glycoprotein or with a human antibody to the sars-cov-2 nucleocapsid protein. sars-cov-2 spike protein (s) expression was detected in vero e6 and ma-104 cell lines that supported sars-cov-2 replication (fig 5a) . the vero e6 and ma-104 cell lines also showed a sars-cov-2 nucleoprotein (np) band, as shown in fig 5b. consistent with the ifa results (fig 4) , viral antigen expression was not detectable in the nonsusceptible sw-13 and hela cell lines (fig 5) . overall, these results showed that the vero e6 and ma 104 cell lines can be efficiently infected by sars-cov-2. to assess the replication kinetics, vero e6 and ma-104 cells were infected with at an moi of 0.1, and the supernatants were harvested at different time points (6, 12, 18, 24, 48 and 72 h post-infection). the vero e6 and ma-104 cell monolayers were then inoculated with 10-fold serial dilutions of the samples. viral titers of the samples were determined by ffa (fig 6a) . the growth kinetics study showed that sars-cov-2 replicated rapidly and efficiently and could be detected within 6 h post-infection in vero e6 and ma-104 cells (fig 6a and 6b ). the western blot assay was performed to examine the production of viral proteins using a rabbit polyclonal antibody to the sars-cov-2 spike glycoprotein (s) (1/1000) (abcam; ab272504) and a human antibody to the sars-cov-2 nucleocapsid protein (np) (1:2500) (genscript; hc2003). 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 1000a, sigma germany). the arrows indicate that the bands at approximately 180 kda ( fig 5a) and 48 kda (fig 5b) sars-cov-2 replicated in vero e6 and ma-104 cells with similar kinetics and achieved similar peak titers of 6.1xlog 6 ffu/ml and 1.6xlog 6 ffu/ml at 48 h of post-infection, respectively ( fig 6a) . however, the viral titers decreased after 48 h infection. at 72 h post-infection, the titers from the samples vero e6 and ma-104 infected with sars-cov-2 were 5.4xlog10 5 ffu/ml and 2xlog10 5 ffu/ml, respectively (fig 6a) . the sequencing produced approximately 4.9 m paired reads (150 bp x2), of which 95.2% of the reads were mapped to the reference genome. after alignment, 99.6% of the genome was covered with a 26200x sequencing depth on average. the whole-genome sequencing of hcov-19/turkey/eragem-001/2020 revealed that the strain had 6 variants, compared to the mn908947.3 reference genome. the detected 2 non-synonymous, 3 synonymous, and 1 utr variants are listed in table 1 . the variants were found to correspond to the genomic positions 1397 and 11083 (orf1ab gene), 23876 (s gene), 26688 (np gene), 29563 (orf 10 gene) and 29742 (3' utr) . the mutations at 1397 and 23876 are non-synonymous, leading to a change from valine to isoleucine in genes orf1ab and s (table 1) . the phylogenetic analysis showed that hcov-19/turkey/eragem-001/2020 was located outside of the main clades (s, g, and v) and clustered with sars-cov-2 isolates from australia, canada, england and kuwait (fig 7) . 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 (g1397a, t28688c, and g29742t) 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 9 sars-cov-2 samples in the nearby clusters had a recent travel history to iran [30] . those samples in nearby clades include several cases from pakistan, kuwait, canada, and norway (fig 7) . the genome sequences of all those cases with a history of travel to iran share three nucleotide substitutions (g1397a, t28688c, and g29742t) in the sars-cov-2 genome, which were also found in the hcov-19/ turkey/eragem-001/2020 strain. the gisaid had only one full genome sequence of sars-cov-2 from iran, which also included the two key mutations (g1397a and g29742t). in addition, analysis of the np partial gene sequences of the iranian samples in gisaid showed that all of the iranian partial sequences (n = 15) also contain the t28688c, which is another key single nucleotide polymorphism (snp) of this clade. our strain also contained g23876a, 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 772nd position, which is located at one of the inner coil motifs of the s protein. another mutation in the orf10 gene (c29563t) 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-2 strains in turkey. the first sequence was generated in march 2020 and deposited in gisaid (epi_isl_417413). the first sequence was not included in the phylogenetic analysis because of its high number of unique mutations (0.44%). however, the sequence comparison showed that both sequences carry key iranian cluster mutations including g1397a, t28688c, and g29742t. the g11083t, g23876a, and c29563t nucleotide changes in our strain were not found in the first sequenced strain in turkey. as of april 2020, a total of 17 sars-cov-2 sequences have been submitted to gisaid samples from turkey. two of those sequences (turkey/hgsm/5516/2020 and turkey/hgsm/8010/2020) were located in the same cluster and share cluster-specific variants (g1397a, t28688c, g29742t). the rest of the submitted strains clustered with several other european clades. sars-cov-2 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 [31] [32] [33] [34] [35] . the sars-cov-2 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-2 in turkey economy [36] [37] [38] . 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-2 was first isolated by using human airway epithelial cells on january 7, 2020 [14, 39] . following to the first isolation of sars-cov-2 in china, several groups have also isolated sars-cov-2 by using the vero cell line [40] [41] [42] [43] . in this study, we isolated the sars-cov-2 from the nasopharyngeal sample of a patient in turkey with confirmed covid-19. isolation of sars-cov-2 was successfully achieved in vero e6 cells in the absence of trypsin. sars-cov-2 caused morphological changes such as rounding, detachment/floating and degeneration (fig 1b, 1c and 1d ). it is essential to define different target cells for sars-cov-2 for further studies on virushost interactions. chu and colleagues identified different cell lines in which both sars-cov and sars-cov-2 replicated efficiently, but the cytopathic effects were only seen in the nonhuman primate kidney cell lines veroe6 and frhk4 [40] . recently, a study showed that the vero e6 and vero ccl81 cell lines were infected with sars-cov-2 and exhibited to sars--cov-2 specific cpe. these authors found that huh7.0 and 293t cells showed only modest viral replication but no cpe was observed, suggesting that both vero cell types support amplification and replication of sars-cov-2 but that vero e6 cells are more suitable for amplification and quantification [42] . in this study, we assessed the susceptibility of vero e6, ma-104, sw-13 and hela cell lines to infection by sars-cov-2. we determined that the vero e6 and ma-104 cell lines were permissive for sars-cov-2 infection. initial cpe formation in vero e6 and ma-104 cell lines infected with sars-cov-2 was observed as early as at 24 h postinfection (figs 1b and 3b, respectively) . immunoblotting analysis also confirmed that only vero e6 and ma-104 cell lines infected with sars-cov-2 showed the expression of the virus specific proteins expression (fig 5) . in addition to vero e6 cells, the ma104 cell line pertaining to its suitability for sars-cov-2 proliferation and facilitate further study of sars-cov-2. our results are in agreement with the previous reports showing that sars-cov replicated efficiently and caused cpe in vero and ma-104 cell lines [44] [45] [46] [47] . in order to evaluate the viral growth kinetics of sars-cov-2, the vero e6 and ma-104 cell lines were infected with at an moi of 0.1. viral replication was assessed at different time points (6, 12, 18, 24, 48 and 72 h post-infection). we used ffa for all virus titration experiments in this study, since ffa is independent of cell death. sars-cov-2 replicated with a similar kinetics in vero e6 and ma-104 cells, but the ma-104 cells supported replication of sars-cov-2 slightly less well than the vero e6 cells (fig 6a) . viral replication could be detected at 12 h post-infection, and continued to increase gradually, peaking at 48 h post-infection (fig 6a and 6b ). the decline in virus titer at 72 h post-infection which might be due to death of the infected cells or to the cell lysis ( fig 6a) . our data are in agreement with that of harcourt et.al., in which sars-cov-2 replicated rapidly in vero cells after an initial eclipse phase and increased gradually, peaking at 48 h post-infection [42] . lastly, we have compared the whole genome of hcov-19/turkey/eragem-001/2020 with a dataset of 3970 available sars-cov-2 complete genomes from different countries retrieved from gisaid. the hcov-19/turkey/eragem-001/2020 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 2020. the cases in the nearby clusters were reported to have a travel history to iran and shared the common unique nucleotide substitutions, g1397a, t28688c, and g29742t, which were also found in our strain and partially in several iranian cases [30] . 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-19/turkey/ eragem-001/2020 has the g23876a 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 (g11083t, g23876a, and c29563t) 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-2 from a patient in turkey with confirmed covid-19. we determined that the vero e6 and ma-104 cell lines might be a good choice of cell culture model for sars-cov-2 that supports viral replication kinetics, development of cpe and subsequent cell death. we also showed that hcov-19/turkey/eragem-001/2020 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. (docx) fields virology. philadelphia continuous and discontinuous rna synthesis in coronaviruses downstream sequences influence the choice between a naturally occurring noncanonical and closely positioned upstream canonical heptameric fusion motif during bovine coronavirus subgenomic mrna synthesis brian bovine coronavirus 5'-proximal genomic acceptor hotspot for discontinuous transcription is 65 nucleotides wide zoonotic origins of human coronaviruses coronavirus pathogenesis emerging coronaviruses: genome structure, replication, and pathogenesis epidemiology, genetic recombination, and pathogenesis of coronaviruses mers coronaviruses in dromedary camels origin and evolution of pathogenic coronaviruses the sars wake-up call sars and mers: recent insights into emerging coronaviruses middle east respiratory syndrome: emergence of a pathogenic human coronavirus a novel coronavirus from patients with pneumonia in china return of the coronavirus: 2019-ncov gisaid database. 2020 coronavirus the new pneumonia "covid-19 a familial cluster of pneumonia associated with the 2019 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 99 cases of 2019 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 7: improvements in performance and usability iq-tree: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies an emergent clade of sars-cov-2 linked to returned travellers from iran an interactive web-based dashboard to track covid-19 in real time characteristics and outcomes of 21 critically ill patients with covid-19 in washington state host and infectivity prediction of wuhan 2019 novel coronavirus using deep learning algorithm first case of 2019 novel coronavirus in the united states the novel zoonotic covid-19 pandemic: an expected global health concern economic impacts of wuhan 2019-ncov on china and the world the socio-economic implications of the coronavirus and covid-19 pandemic: a review multidisciplinary research priorities for the covid-19 pandemic: a call for action for mental health science. lancet psychiatry who. novel coronavirus(2019-ncov) situation report-1 virus isolation from the first patient with sars-cov-2 in korea serological and molecular findings during sars-cov-2 infection: the first case study in finland severe acute respiratory syndrome coronavirus 2 from patient with 2019 novel coronavirus disease, united states. emerg infect dis inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2. cell comparative tropism, replication kinetics, and cell damage profiling of sars-cov-2 and sars-cov and implications for clinical manifestations, transmissibility, and laboratory studies of covid-19: an observational study sars-associated coronavirus replication in cell lines severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis exogenous ace2 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-001572-ap4ro5me 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 1 date: 2015-02-28 journal: biomed res int doi: 10.1155/2015/358462 sha: doc_id: 1572 cord_uid: ap4ro5me 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 1 was serially passaged on five human cell lines, hl60, k562, kg1, thp-1, and u937. sabin type 1 was capable of efficiently replicating in three cell lines (k562, kg1, and u937), yielding high viral titers after replication. expression of cd155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed cd155. furthermore, we showed that passaged virus replicated more efficiently than parental virus in kg1 cells, yielding higher virus titers in the supernatant early after infection. infection of cell lines at an moi of 0.01 resulted in high viral titers in the supernatant at day 4. infection of k562 with passaged sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. altogether, these data suggest that k562, kg1, and u937 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-5 and wi-38 [1, 2] . 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 [3] [4] [5] . 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. [6] ). 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 2 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 [7] . 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 140 to 165 are not tumorigenic in immunocompromised mice [8] [9] [10] 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 185 [11] . 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 [12] , meaning that vero cells can be used for the production of a number of different vaccines [13] [14] [15] [16] . 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 [17] . 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 2012 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/ucm319573.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 [18] , hela cell line [19, 20] , and the per.c6 cell line of which the latter was immortalized by transfection with adenoviral e1 proteins [21] . 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 [22] [23] [24] [25] [26] . 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 30 years with respect to the risks and benefits of immortalized tumorigenic cell lines [7, 26] . 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 [27] . 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, hl60, k562, kg1, thp-1, and u937 [28] [29] [30] [31] [32] . 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 [33] [34] [35] . possibly, the cells can become infected as progenitor cells, whereas infection is not possible when the cells are differentiated or vice versa. interestingly, k562 cells are currently used as a vaccine for patients with lung cancer. irradiated k562 cells, transfected with the gene encoding gm-csf and cd40 ligand, were mixed with allogeneic tumor cells and this vaccine was tested in a phase ii trial in lung cancer patients [36] . 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 hl60, u937, k562, kg1, and thp-1 were cultured in imdm (hl60, u937, k562, and kg1) (invitrogen) or rpmi (thp-1) supplemented with 10% fetal bovine serum (fbs, paa) and 10 u/ml penicillinstreptomycin (gibco) at 37 ∘ c in a 5% co 2 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 2 mm glutamine (life technologies) at 37 ∘ c in a 5% co 2 humidified atmosphere. cho suspension cells, which served as a negative control, were cultured in ex-cell 302 medium (sigma) in shaker flasks (corning) gently rotating at 50-100 rpm at 37 ∘ c in a 5% co 2 humidified atmosphere. to determine whether k562 cells were capable of growing in bioreactors, cultibags (sartorius stedim) were inoculated with cells at a concentration of 0.3 × 10 6 cells/ml in 1 l of culture medium. cultibag cultures were performed at 37 ∘ c, dissolved oxygen concentration of 50%, ph 7.2 at a constant rocking speed of 10 rpm, and an angle of 7 ∘ . tumor cell lines. to serially passage sabin poliovirus type 1 on the hematopoietic cell lines, 1 × 10 6 cells were infected biomed research international 3 in t25 flasks with an moi of 1. cells and supernatants were harvested if full cytopathic effect (cpe) was observed or after 1 week of culture. after freeze-thawing three times to lyse the cells, samples were centrifuged at 3000 rpm to remove cellular debris and half of the supernatant was used to reinfect new hematopoietic cells. this was repeated for 4 times to allow the virus to adapt to the new cell lines in 5 passages. in the last infection round, 1 × 10 7 cells in a t175 flask were infected and samples were harvested at day 3 (vero and u937) or day 6 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 1 from the parental virus or virus that was passaged for 5 times on the hematopoietic cell lines at moi 1 or moi 0.01, and samples of the supernatant and cellular lysates were harvested at different time points. after harvesting, the supernatant was centrifuged at 1000 rpm for 4 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 1000 rpm, pbs was removed and the cell pellet was resuspended in 1 ml of pbs. the cellular samples were freeze-thawed three times to release virus from the cells, and after centrifugation at 3000 rpm supernatant was transferred to a new tube. the virus titer in all the samples was determined by end-point titration on vero cells. k562 cells grown in cultibags were infected at an moi of 0.01 with passaged sabin poliovirus type 1 when cells reached a concentration of 1.2-1.5 ×10 6 cells/ml, and samples of the culture medium were taken at days 3, 4, 5, and 6 after infection. samples were filtered (0.22 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 1 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 1 × 10 4 cells/100 l in 96-well flat bottom plates in m199 medium containing 10% serum. serial 10-fold dilutions were prepared from cellular lysates or supernatant from hematopoietic cells infected with sabin poliovirus type 1 in serum-containing m199 medium, and 50 l of these dilutions was added to 6 separate wells. after 7 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 50% cell culture infective dose (ccid50)/ml. to calculate the plaque forming unit titer (pfu) from the ccdi50 value, the ccid50 was multiplied with 0.69 to generate the pfu titer/ml. to be able to compare the number of infectious sabin type 1 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 [37] . briefly, plates were coated with an anti-sabin type 1 caprylated bovine antiserum diluted 1 : 1600 in pbs (gibco) overnight at 4 ∘ c. after washing, samples were added for 30 minutes at 37 ∘ c. after washing, a mixture of a sabin type 1 specific mouse monoclonal antibody and an hrp-labeled goat anti-mouse antibody were added for 30 minutes at 37 ∘ 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 cd155 (ebioscience), cd54 (bd pharmingen), and car (millipore) and fitc-labeled anti-human cd81 (bd pharmingen) were used for flow cytometric analyses, with fitc-or pe-labeled igg1 antibodies (bd pharmingen) as controls. antibody staining was performed in pbs supplemented with 0.1% bsa and 0.02% sodium-azide for 30 min at 4 ∘ 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 1, cells were infected with an moi of 1 and cells together with supernatant were harvested at day 3 (for all virus passages in vero cells and for passages 3-5 on u937 cells) or day 6 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 4 more times, and virus derived from passage 5 was used for additional experiments. in the samples derived from all 5 passages, the virus titer (ccid50/ml) was determined by a virus titration assay. as shown in figure 1(a) , replication was efficient in the control cell line vero, grown in serum-free medium, yielding high titers of more than 1 × 10 7 ccid50/ml in all 5 passages. in cho cells, sabin poliovirus type 1 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 2-5 of sabin poliovirus type 1 added to cho cells, as expected no virus could be detected. a comparable pattern was observed after infection of thp-1 and hl60 cells with sabin poliovirus type 1, suggesting that these two cell lines were refractory for the virus. the k562 and u937 cell lines were highly permissive for sabin poliovirus type 1, resulting in high virus titers in all 5 viral passages. kg1 cells were also fully permissive for sabin poliovirus type 1, but the amount of virus obtained after infection of kg1 cells was slightly lower compared to sabin poliovirus type 1 that replicated in the other permissive cell lines. microscopically it was observed that u937 cells infected with virus from passages 0-2 did not show clear cytopathic effects (cpe) after 6 days of culture, whereas cells infected with virus from passages 3, 4, and 5 were harvested after 3 days because full cpe was observed. a photographic overview of cells infected with virus from passage 4 is shown in figure 1(b) . also, in k562 cells, clear cpe was visible at day 6 after infection. the amount of virus produced after replication of sabin poliovirus type 1 in k562 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 1(c) . for vero cell derived sabin poliovirus type 1, the d-antigen per infectious virus particle was 4 du/10 7 ccid50. 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 1.2 and 2.3 du/10 7 ccid50 depending on the cell line used. hematopoietic cell lines correlated with the capacity to propagate sabin poliovirus type 1, human cd155 expression was determined using facs analysis. in figure 2 it can be seen that all hematopoietic cell lines, as well as vero cells, express cd155. vero, u937, and k562 had the highest level of expression, whereas the expression level of thp-1, kg1, and hl60 cells was lower. also, not all hl60 cells were positive for cd155. since sabin poliovirus type 1 was not capable of replicating in thp1 and hl60 cells whereas replication in kg1 cells was efficient, expression levels of cd155 thus cannot fully predict the capacity of sabin poliovirus type 1 to replicate in these cell lines. supernatant at day 1 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 2 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 2 in the supernatant is comparable to the virus titer obtained 2 days after replication of sabin poliovirus type 1 in vero cells. also, the virus titer in the culture medium remained stable during the 7 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 k562 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 0.01 of passaged sabin poliovirus type 1, and supernatant and cellular lysates were harvested separately at day 4 and day 7. the total viral titer in these samples was determined and is shown in figure 4 . in the supernatant of all cell lines tested, at day 4, a high virus titer, comparable to sabin poliovirus type 1 replicated in vero cells, was observed in the culture medium, indicating that virus replication was efficient during multiple rounds of replication. in k562 cells, the virus titer was moderately further increased at day 7 after infection, whereas in the other cell lines the virus titer at day 7 in the culture medium slightly decreased compared to the titer at day 4. interestingly, at both day 4 and day 7 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 4 or day 7 by sabin poliovirus type 1. sabin poliovirus type 1. to determine whether it is possible to produce sabin poliovirus type 1 at a larger scale, k562 cells were grown in cultibags. cells were inoculated at a concentration of 0.3 × 10 6 cells/ml in 1 l culture medium. after 4 days, in which the k562 cells grew to a concentration of 1.2-1.4 × 10 6 cells/ml, the cells were infected with the passaged sabin poliovirus type 1 at an moi of 0.01. at days 3, 4, 5, and 6, 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 5 (a), the virus replicated efficiently, resulting in titers of 1 × 10 9 ccid50/ml in the culture medium already at day 3 after infection, which remained stable for at least the following 3 days. the d-antigen level at day 3 after infection ( figure 5(b) ) was somewhat lower than that at later time points. also, a large variation in the d-antigen level between the 3 experiments was observed at day 3, suggesting that not all viruses expressed d-antigen yet. however, at days 4, 5, and 6 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 cd54, car, and cd81 was performed. cd55, car, and cd81 are the receptors for rhinovirus, coxsackie, adenovirus, and hepatitis c virus, respectively. in figure 6 , the percentage of cells expressing the specific receptor is shown. all cell lines, except hl60, highly expressed cd54 and cd81. hl60 cells did express high levels of cd81, but cd54 was only expressed by 50% of the cells. with respect to car expression, differences between the cell lines were observed. a minority of k562 cells expressed car, whereas, for the other cell lines tested, 50-90% 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.) [38] . among the greatest success stories are the eradication of smallpox virus, rinderpest, and the polio eradication program, where poliovirus type 2 was already eradicated in 1999 [39] [40] [41] . 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.) [42] . 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.) [7] , 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 [43] [44] [45] , it was decided to perform this study with sabin poliovirus type 1 as the first model virus. first, it was determined whether the five selected cell lines were susceptible for poliovirus infection. k562, kg1, and u937 cell lines appeared to be capable of supporting replication of sabin poliovirus type 1, whereas hl60 and thp-1 were not. all five cell lines did express cd155, 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 cd155 expressing hl60 and thp-1 cells could be that (i) cd155 expressed on these cell lines is nonfunctional, (ii) the nonsusceptible cell lines lack the expression of a coreceptor on the cellular membrane, or (iii) hl60 and thp-1 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 [33] [34] [35] , it would also be interesting to determine permissiveness of hl60 and thp-1 for sabin type 1 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 [46] , 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, k562 cells were the least differentiated, but with respect to viral replication we did not observe a difference between k562 cells and the more differentiated cell lines. another study compared the replication capacity of mahoney poliovirus on k562 and u937 cells to hela cells [47] . compared to hela cells, poliovirus replicated less efficiently in both k562 and u937 cells yielding a 20-fold and a 50-fold reduced virus output. finally, a study by benton et al. showed differences between k562 clones in their response to poliovirus infection, because two out of four k562 cell lines were killed by the poliovirus, whereas in the two other cell lines persistent infections were established [48] . in our study, we did not observe a persistent infection of k562 cells, nor that the poliovirus replicated less efficiently in u937 cells or the less-differentiated k562 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 1 were studied, meaning that the virus has had 5 passages to adapt to the new cell line. we did, however, observe already after the first passage of sabin type 1 in k562 or u937 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 1 poliovirus, and in our study attenuated sabin poliovirus type 1 was used. in two more recent papers, the replication capacity of sabin types 1, 2, and 3 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 1, 2, and 3. of the 5 cell lines tested (bhk-21, cho-k1, cap, smdck, and age1.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 [49] . in the second study, published by sanders et al., the capacity of per.c6 to support replication of brunenders, mef-1, and saukett poliovirus was determined in comparison to vero cells [50] . per.c6 is an immortalized human cell line capable of supporting the replication of a number of viruses, like influenza virus and west nile virus [51, 52] . the poliovirus was capable of replicating efficiently in the per.c6 cell line, resulting in a high virus yield, which can be attributed to the fact that per.c6 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 [49] . 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 k562 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 kg1 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 k562, kg1, and u937 cell lines are efficient in supporting the replication of sabin poliovirus type 1. all five human hematopoietic cell lines expressed cd155, which thus did not explain susceptibility to viral replication, although we did not study the functionality of cd155. furthermore, we have shown that in kg1 cells the passaged sabin poliovirus type 1 replicated more rapidly than the control virus. infection of k562, kg1, and u937 at an moi of 0.01 resulted in a high viral titer in the culture medium after 4 days. also, k562 cells grown and infected with adapted sabin poliovirus type 1 in a disposable bioreactor system yielded high viral titers in the culture medium. altogether, it is concluded that k562, kg1, and u937 cell lines can be used for the propagation of poliovirus and in follow-up studies the capacity of other (entero)viruses to 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key: cord-351377-xorj8tnz 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 52 strain date: 2018-10-04 journal: viruses doi: 10.3390/v10100543 sha: doc_id: 351377 cord_uid: xorj8tnz 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 2b (g2b) pedv taiwan pintung 52 (pedvpt) strain, pedvpt-p96, and determined its promising host immune response against the virulent pedvpt-p5 strain. to study the attenuation determinants of pedvpt-p96 and establish a pedvpt-p96-based recombinant vector as a vaccine platform for further antigenicity modification, ipedvpt-p96, a full-length cdna clone of pedvpt-p96, was established. comparing to the parental pedvpt-p96 virus, the ipedvpt-p96 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 5-week-old piglet model, fecal viral shedding was observed in the pedvpt-p96-inoculated piglets, whereas those inoculated with ipedvpt-p96 showed neither detectable fecal viral shedding nor pedv-associated clinical signs. moreover, inoculation with ipedvpt-p96 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-p5 challenge compared to the parental pedvpt-p96. in the present study, an infectious cdna clone of an attenuated g2b pedv strain was successfully generated for the first time, and the in vitro and in vivo data indicate that ipedvpt-p96 is further attenuated but remains immunogenic compared to its parental pedvpt-p96 viral stock. the successful development of the ipedvpt-p96 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 [1] . the genome of pedv is about 28 kilobase pairs in length and comprises of seven open reading frames (orf), including orf1a and b genes that constitute the 5 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 orf3 gene with an undetermined function [2] . 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 [1] . in late 2010, new and highly virulent pedv strains arose in china and spread rapidly worldwide by late 2013, resulting in nearly 100% mortality in the affected nursing piglets [3] [4] [5] . to date, there are still indelible endemics and considerable economic losses in the global swine market [6] . besides, the protection conferred by currently available vaccines is, unfortunately, unsatisfactory [6, 7] . based on the nucleotide identity of the s gene, pedvs are categorized into four genogroups (gs), namely g1a, g1b, g2a, and g2b [8] . among these, the g2b pedv strains that predominate the field in asia and north america, show higher pathogenicity [9] and appear to elicit broader protection across different genogroups [10] [11] [12] . 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 [13, 14] , the detailed mechanism remains elusive. previously, we generated an attenuated g2b taiwan pedv strain, pedv pintung 52 passage 96 (pedvpt-p96) virus, by serial passage of the parental pedvpt strain in vero cells [15] . despite the high potential of pedvpt-p96 as a future vaccine candidate against pedv as indicated by its reduced pathogenicity and robust host immune response in our 5-week-old piglet model [15] , the difficulty in pedv isolation and subsequent lengthy passage process rendered the pedvpt-p96 unable to promptly respond to the vast outbreak in late 2013. this year, zhou et al. [16] reported a new disastrous swine disease outbreak in china in 2016 caused by an hku2-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 [17] [18] [19] [20] [21] . 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-p96, ipedvpt-p96, was generated. in addition, the pathogenicity, immunogenicity, and protection against virulent pedvpt-p5 challenge by ipedvpt-p96 were evaluated in the 5-week-old piglet model. our data suggest that the ipedvpt-p96 virus was more attenuated but able to elicit similar immunogenicity and immunoprotection against the autologous virulent pedvpt-p5 challenge compared to the parental pedvpt-p96 virus. this ipedvpt-p96 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 c1008 cells (atcc no. crl-1586) were maintained in dulbecco's modified eagle's medium (dmem, gibco, grand island, ny, usa) supplemented with 10% fetal bovine protein (fbs), 250 ng/ml amphotericin b, 100 u/ml penicillin, and 100 µg/ml streptomycin. viral stock of pedvpt-p96 in post-inoculation medium (pi medium) containing dmem supplemented with 0.3% tryptose phosphate broth (tbp), 0.02% yeast extract (0.02%), and 10 µg/ml trypsin, as prepared in our previous study [15] , was used for generating the infectious cdna clone and as the control for in vivo and in vitro studies, whereas the virulent pedvpt-p5 virus was used for animal challenge. the strategy used to engineer the full-length cdna clone of pedvpt-p96, namely ipedvpt-p96, was modified according to a previously published method [22] . briefly, the complete genome of pedvpt-p96 (genbank accession no. ky929406) was divided into six fragments by pcr amplification using primer pairs (table 1 ) incorporated with specific type-iis restriction enzyme sites for seamless ligation. fragment a contained a t7 promoter sequence at its 5 end to allow in vitro transcription and the sequence was designed following the article published previously [19] ; a 25-adenosine sequence was added to the 3 end of fragment f to simulate polyadenylation. a naturally occurring bsai site in fragment e was removed by introducing a silent mutation (c24341t) by site-directed mutagenesis according to the previously described protocol [23] . an additional synonymous mutation (t24841c) 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 (pjet1.2; thermo fisher scientific, waltham, ma, usa). each subclone was digested with the corresponding type-iis restriction enzymes as indicated in figure 1 and gel-purified using a qiaquick gel extraction kit (qiagen, hilden, germany). assembly of the full-length cdna was conducted by employing t4 ligase (neb, ipswich, ma, usa) overnight at 4 • c. the ligated full-length cdna was phenol-chloroform extracted and in vitro transcribed to full-length rna transcripts using a mmessage mmachine t7 transcription kit (ambion, austin, ca, usa) following the manufacturer's instructions. the cap analog to gtp ratio was adjusted to 1:1 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 t7 promoter sequence and poly-a tail at the 3 and 5 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 (30 µl) and 5 µg of n transcripts were mixed thoroughly and electroporated into vero cells in a suspension of 800 µl with 10 7 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 450 v, 50 µf and 2-3 s rest between each pulse. after electroporation, the cells were initially incubated at room temperature for 15 min and then resuspended in dmem supplemented with 10% fbs in a 75-cm 2 flask overnight at 37 • 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 2-3 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-p96 for further use. the viral stock was titrated in vero cells in a 96-well plate to determine viral titer. to detect the pedv antigen, immunocytochemistry (icc) was performed as described previously [15] . briefly, vero cells infected with ipedvpt-p96 showing typical cpe were fixed with 80% ice-cold acetone, air-dried, and incubated with an in-house anti-pedv n antibody at room temperature (rt) for 1 h. after washing three times with pbs, a polyclonal anti-rabbit/mouse immunoglobulin, envision-dab + system (dako, carpinteria, ca, usa), was applied for 1 h at rt. following three washes with pbs, the cells were incubated with 3, 3 -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 [15] and a primer pair (sf5 and n-4 listed in table 1 ) targeting the genome between nucleotides 23037 to 25574 was used to identify the presence of marker mutations, c24341t and t24841c, in the ipedvpt-p96 viral stock. the growth characteristics of ipedvpt-p96 and pedvpt-p96 in vero cells were evaluated and compared by examining the growth kinetics and plaque morphologies. confluent monolayers of vero cells were prepared on 6-well plates and infected with each virus at the multiplicity of infection (moi) 0.01 for 1 h at 37 • 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, 0, 12, 24, and 36 h post-inoculation (hpi), were collected and subjected to titration in vero cells seeded in 96-well plates. plaque assays were performed to characterize the plaque morphologies. after the adsorption of pedvs at moi 0.01, vero cells were washed twice with dpbs and overlaid with pi medium containing 1% agarose (invitrogen, carlsbad, usa) pre-warmed to 42 • c. upon solidification of the overlays, the plates were incubated for 3 days at 37 • c to allow pedv-infected vero cells to produce distinct plaques. the cells were fixed in 3.16% neutral formalin for 1 h at rt. the semisolid overlays were then removed manually and the cells were stained with 1% crystal violet in 20% ethanol and distilled water for 1 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, 4-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 g2b pedv strain infection. these piglets were randomly assigned to three groups, including the pedvpt-p96 group (n = 5), ipedvpt-p96 group (n = 5), and mock group (n = 5), and acclimated for one week prior to inoculation. at 5 weeks of age, the piglets in each group were orally inoculated with 4 ml of 5 × 10 5 tcid 50 /ml of pedvpt-p96, 5 × 10 5 tcid 50 /ml of the pedvpt-p96 virus, or pi medium, respectively. to evaluate the protective efficacy induced by ipedvpt-p96, piglets at 9 weeks of age in all groups were orally challenged with 5 ml 10 5 tcid 50 /ml of pedvpt-p5. 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.: ntu105el-00160. fecal consistency was monitored daily and scored visually as 0 = normal, 1 = loose, 2 = semi-fluid, and 3 = watery, as described previously [15] . 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 1000 µl dpbs, pulse-vortexed for 5 s and precipitated by centrifugation at 13,000 rpm for 5 min. rna was extracted automatically from 200 µl of stool suspension on a qiacube using the cador pathogen 96 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 [4] and a quantinova ® probe pcr kit (qiagen, hilden, germany) on a cfx96 thermal cycler (bio-rad, hercules, ca, usa). the thermal profile comprised an initial denaturation at 95 • c for 2 min followed by 45 cycles of 95 • c for 15 s followed by 60 • c for 15 s. the detection limit of the assay was determined by generating standard curves from serial 10-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 100 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 [24] . in brief, 96-well, flat-bottom microtiter plates (nunc, roskilde, denmark) were coated with 2 µg/ml purified recombinant pedvpt s protein (200 ng/well) and incubated overnight at 4 • c. the plates were washed six times with 100 µl of pbst (pbs containing 0.05% tween 20) and then blocked with 300 µl of blocking buffer (1% bovine serum albumin in pbs) at rt for 1 h. for the detection of plasma igg, 100 µl of 40-fold diluted plasma samples in blocking buffer were added following six washes and incubated at rt for 1 h. for fecal and salivary iga, 100 µl of eluted fecal suspension and saliva at 1:2 dilution in blocking buffer were added following six washes and kept overnight at 4 • c. after incubation, the samples were discarded and the plates were washed six times. to detect plasma igg, and the fecal and salivary iga, 100 µl of either horseradish peroxidase (hrp) conjugated goat anti-pig igg (kirkegaard & perry laboratories, milford, ma, usa) at 1:1000 dilution, or goat-anti-pig iga (abcam, cambridge, uk) diluted 1:10,000 were added, respectively, and incubated at rt for 1 h. following a wash step, 50 µl of tetramethylbenzidine (tmb) substrate solution (kirkegaard and perry laboratories) was added to allow color development at rt for 10 min. the reactions were terminated by adding 50 µl of tmb stop solution (kirkegaard and perry laboratories) to each well. the optical density (od) at 405 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 56 • c for 30 min to inactivate complement prior to use. for each well, mixtures containing 50 µl of pedvpt-p5 virus (50 viral particles) and 50 µl of 2-fold diluted plasma samples in pi medium were incubated at 37 • c for 1 h before applying to vero cells (2 × 10 4 /well). after incubation with the virus-plasma mixture for 1 h, vero cells were washed twice and maintained in pi medium for 24 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 6.0 (graphpad software, san diego, ca, usa) with two-way anova by time. a p value less than 0.05 was considered statistically significant. to generate the full-length cdna clone of ipedvpt-p96, the complete genome sequence of pedvpt-p96 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-p96 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 2a) . the presence of ipedvpt-p96 was confirmed by the detection of the pedv n protein by immunocytochemistry ( figure 2a ) and sequence analyses for identification of the introduced substitutions, c24341t and t24841c ( figure 2b ). after an additional day, recombinant viral supernatants were harvested when the cpe reached 90% of the confluent monolayer. an ipedvpt-p96 viral stock with a titer of 5.62 × 10 6 tcid 50 /ml was prepared by an additional passage of the viral supernatants in the vero cells. to characterize the recombinant ipedvpt-p96, plaque morphologies and growth kinetics of the recombinant ipedvpt-p96 in vero cells were compared to those of the parental pedvpt-p96 virus. interestingly, aside from the constantly short diameter, the plaque size of ipedvpt-p96 appeared to be more uniform than that of pedvpt-p96 ( figure 2c ). as to the replicative capacity, the multistep growth kinetics of both the ipedvpt-p96 and pedvpt-p96 viruses in vero cells were examined. at moi of 0.1, while pedvpt-p96 replicated rapidly to the titer of 4.58 ± 0.14 tcid 50 /ml at 12 hpi, reached the peak viral titer of 6.42 ± 0.29 tcid 50 /ml at 24 hpi, and showed a declined viral titer of 5.58 ± 0.38 tcid 50 /ml at 36 hpi. ipedvpt-p96 showed a steady increase in viral progenies and a significantly decreased viral titer of 3.58 ± 0.14 tcid 50 /ml at 12 hpi, a comparable viral titer of 6.16 ± 0.38 tcid 50 /ml at 24 hpi, and a significantly high viral titer of 7.16 ± 0.38 tcid 50 /ml at 36 hpi ( figure 2d ). at mois of 0.001 and 0.00001, similar to those observed at 0.01 moi, ipedvpt-p96 replicated slower in the beginning, reached a similar peak viral titer as compared to pedvpt-p96 and showed a relatively slower reduction of viral titer after reaching the peak titer (see figure s1 ). to evaluate the pathogenicity of the ipedvpt-p96 virus, 5-week-old crossbred piglets were orally inoculated with 4 ml of 5 × 10 5 tcid 50 /ml of the ipedvpt-p96 virus, pedvpt-p96 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 3 ). while one piglet in the pedvpt-p96 group showed intermittent loose diarrhea (score = 1) and viral shedding at 6 to 11 days post-inoculation (dpi) with the peak viral titer of 1.45 ± 3.24 log 10 rna copies/ml at 8 dpi (figure 4) , no evidence of pedv-associated clinical signs and fecal viral shedding were demonstrated in both ipedvpt-p96 and mock groups. after the oral inoculation of piglets with the ipedvpt-p96 and pedvpt-p96 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 14 dpi and sustained until 28 dpi in piglets from both the ipedvpt-p96 and pedvpt-p96 groups ( figure 5) . moreover, subtle increases in the mean s/p ratios of fecal and salivary pedv-specific iga were also observed in both the ipedvpt-p96 and pedvpt-p96 groups compared to those of the mock group at 28 dpi ( figure 6 ). after oral inoculation with the virulent pedvpt-p5 virus, the daily fecal viral sheddings and fecal consistencies in all groups were evaluated. similar to our previous data [15] , all piglets in the mock group showed the differing severity of clinical signs for diarrhea at 3-6 days post-challenge (dpc) (figure 7) . the mean value of the pedv rna copies in feces of the mock group became detectable at 3 dpc, shot up to a peak titer of 5.72 ± 3.62 log 10 rna copies/ml at 5 dpc, and then declined to a constantly low viral load of approximately 1 log 10 rna copies/ml at 9-16 dpc. in the pedvpt-p96 group, two piglets showed loose diarrhea (score = 1) for one day at 6 and 7 dpc, respectively. the onset of fecal viral shedding in the pedvpt-p96 group after the challenge was further delayed to 6 dpc compared to the other two groups. the highest viral titer of the pedvpt-p96 group also appeared at 6 dpc, calculated as 3.66 ± 3.41 log 10 rna copies/ml. in the ipedvpt-p96 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-p96 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 2.75 ± 3.77 to log 10 rna copies/ml to undetectable levels ( figure 5 ). 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-p5 challenge, after which the mean s/p value rose to 0.75 ± 0.43 at 14 dpc ( figure 5 ). on the other hand, the mean values of systemic pedv-specific igg in blood from the ipedvpt-p96 and pedvpt-p96 groups increased sharply to 1.54 ± 0.74 and 1.43 ± 0.62 at 14 dpc, respectively, which was significantly higher than that of the mock group ( figure 6 ). 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-p96 and pedvpt-p96 groups after challenge ( figure 6 ). among them, the s/p values of salivary anti-pedv specific iga, 0.5 ± 0.33 and 0.37 ± 0.22, in the ipedvpt-p96 and pedvpt-p96 groups, respectively, were significantly higher than those of the mock group (0.13 ± 0.08) at 14 dpc (figure 7) . the vn antibody titers of piglets in each group are depicted in figure 7 . similar to the trend observed in systemic plasma igg titers, the ipedvpt-p96 group showed a comparable vn antibody titer against pedvpt-p5 to that of the pedvpt-p96 group through the study. the mean vn antibody titers of both groups increased mildly at 28 dpc and spectacularly at 14 dpc and were higher than those of the mock group during the study (figure 8 ). 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-p96, and evaluated it's in vitro and in vivo characteristics. compared to the parental pedvpt-p96 virus, ipedvpt-p96 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-p96 is less complex than that in the original pedvpt-p96 stock. moreover, neither fecal pedv rna shedding nor a pedv-associated clinical illness was detected in conventional 5-week-old piglets inoculated with the ipedvpt-p96 virus, indicating a further attenuated phenotype in vivo. importantly, piglets in the ipedvpt-p96-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-p5 challenge compared to the parental pedvpt-p96-inoculated piglets. taken together, our results suggest that ipedvpt-p96 is immunogenic in piglets and could be a potential safe viral vector candidate for vaccine development. while inoculation with ipedvpt-p96 was demonstrated to completely protect the piglets from developing diarrhea after challenge with the virulent pedvpt-p5, the ipedvpt-p96-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-p96-inoculated group. these data suggested that ipedvpt-p96 conferred a relatively weakened protection than that of the parental pedvpt-p96. considering that the major variation between the pedvpt-p96 and ipedvpt-p96 viruses should be the heterogeneity of viral population as noted in plaque assays wherein the ipedvpt-p96 virus produced more uniform plaques than those of the parental pedvpt-p96 virus in vero cells, we speculate that the decrease in quasispecies diversity in ipedvpt-p96 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-p96 generated by sanger sequence (see table s1 ). the results clearly demonstrated that the pedvpt-p96 carried a greater sequence diversity than that of the ipedvpt-p96, including 23 single nucleotide variants (snv) and resultant 16 amino acid substitutions; of interest, seven snv were found in the spike gene and 3 of them were the same as the virulent pedvpt-p5. for ipedvpt-p96, excluding the artificially introduced marker mutations and those derived from the snv of pedvpt-p96 upon the initial construction of subclones of ipedvpt-p96, only 3 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 [25, 26] . 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 [27, 28] , it is possible that the consensus sequence used for constructing ipedvpt-p96 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-p96 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-p96-inoculated group. this speculation could be tested by comparing the tissue tropism and the quantity of pedv antigens of both pedvpt-p96 and ipedvpt-p96 in enterocytes using a 7-day-old piglet model. other explanation for the attenuation of ipedvpt-p96 might be attributed to the possible addition of the five nucleotides "ggaga" at the very extreme of the 5 utr based on our primer design (see table 1 ). considering the location that these additional nucleotides sequence should neither alter the secondary structures of the sl1 stem-loop, that serve as cis-acting elements required for driving subgenomic rna synthesis nor change any consensus transcription regulatory sequences 5 -xua(a/g)ac-3 , we assumed that the effect of these additional nucleotides in the rescued ipedvpt-p96 virus on replication might be minimal. nonetheless, further studies are required to determine the potential effect of these five additional nucleotides on ipedvpt-p96 replication. in the present study, the profile of fecal pedv rna shedding and the pattern of antibody responses after inoculation with pedvpt-p96 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 [15] . that is, it seemed that the conventional piglets used in the current study were more resistant to the pedvpt-p96 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 [9] . 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 21st 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 [29, 30] . in addition, the insertion of other antigens by replacing the accessory orf3 gene or creating a novel expression cassette is also an attractive approach to design multivalent vaccines [17, 18, 21] . under this concept, ipedvpt-p96 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-p96 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 [31] . 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 [30] . accordingly, further characterization of the virulence variation and genetic stability of ipedvpt-p96 after different genetic modifications needs to be conducted. in this article, we described the first successful construction of an attenuated g2b pedv infectious cdna clone of the ipedvpt-p96 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 5 nucleotides at the 5 end of ipedvpt-p96 may contribute to a further attenuated phenotype and potentially less effective immunoprotection against the virulent strain challenge. based on the results of antibody response, a prime-boost strategy or further optimization of ipedvpt-p96 antigenicity is essential to induce sufficient protective immunity in all recipients. together, the full-length cdna clone of ipedvpt-p96 generated herein is expected to provide an access to study the attenuation determinants of pedvpt-p96 and establish a pedvpt-p96-based recombinant vector as a vaccine platform for developing multivalent vaccines for pedv and other porcine pathogens. a new coronavirus-like particle associated with diarrhea in swine genome organization of porcine epidemic diarrhoea virus new variants 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gene expression in mammalian cells coronaviruses as vectors: stability of foreign gene expression recombination in large rna viruses: coronaviruses. seminars in virology the authors would like to thank center for biotechnology, national taiwan university for next generation sequencing and bioinformatics and biostatistics core lab, center of genomic and precision medicine, national taiwan 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-023871-9vi0m378 authors: mizutani, tetsuya title: signaling pathways of sars-cov in vitro and in vivo date: 2009-07-22 journal: molecular biology of the sars-coronavirus doi: 10.1007/978-3-642-03683-5_19 sha: doc_id: 23871 cord_uid: 9vi0m378 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 e6 cells after sars-cov infection. from sars-cov infection to cell death, p38 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 3 (stat3) and p90 ribosomal s6 kinase (p90rsk) are downstream of p38 mapk. akt and jnk (jun nh(2)-terminal kinase) signaling pathways are important to establish persistent infection of sars-cov in vero e6 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 2002 and subsequently spread to 29 other countries, thus emerging as the first nearpandemic infectious disease of the twenty-first century. a worldwide total of 8,096 cases of sars, of which 774 (9.6%) resulted in death, was reported by the world health organization (who) (http://www.who.int/csr/sars/country/table2004_04_ 21/en/index.html). sars-cov belongs to the coronaviridae family (order nidovirales) of enveloped single-stranded positive rna viruses (marra et al. 2003; rota et al. 2003; thiel et al. 2003) . the sars-cov genome is approximately 30 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 5 0 and 3 0 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 60% 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 5 0 leader sequence of approximately 70 nucleotides and poly-a tails at the 3 0 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 3 0 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 70 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 14 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 (1a and 1b) encoded by approximately 60% of the sars-cov viral genome are processed into 16 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. 19 .1). 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 e6 cells are used (mizutani et al. 2004c) . various signaling pathways are activated during the entire process of viral infection, from s protein-ace2 (angiotensinconverting enzyme-2) binding for internalization into the host cells to apoptotic cell death (mizutani 2007) . the most common signaling pathways are mitogen-activated protein kinase (mapk) pathways, which include jun nh 2 -terminal kinase (jnk), extracellular signal-regulated kinase (erk), and p38 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 1999) . 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 p38 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 p38 map kinase is expressed in response to stressors, and viral infection generally induces activation of p38 mapk. the roles of p38 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 p38 mapk. there are at least four isoforms of p38 mapk: p38a, p38b, p38g, and p38d _ 1999; platanias 2003; lee et al. 2004 ), 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 70% similarity at the amino acid sequence level, and their functions are inhibited by the pyridinyl imidazole inhibitor sb203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) imidazole]. conversely, p38g and p38d mapks, which have 60% similarity to p38a, are not inhibited by sb203580. furthermore, p38a and p38b mapks are widely expressed in tissues, whereas the expression of p38g and p38d mapks is tissue-specific. in the field of virology, because sb203580 is generally used as an inhibitor of the p38 mapk signaling pathway, it can be used in studying the role of p38a and/or p38b mapks in sars-cov infection. the kinases upstream of p38 mapk are mkk3 (mapk kinase 3) and mkk6 (mapk kinase 6), which are known to phosphorylate and activate p38 mapk. interestingly, mkk6 has affinity to pkr in the presence of double-stranded rna, poly(ri;rc) and pkr is able to activate mkk6, but not mkk3. this result indicated that interaction of mkk6 and pkr provides a mechanism to regulate activation of p38 mapk (silva et al. 2004) . in hepatitis c virus-core-expressing cells, pkr has an important role in cell cycle arrest and was shown to interact strongly with p38 mapk (spaziani et al. 2006) . as upstream of p38mapk, transforming growth factor (tgf)-b-activating kinase (tak1), apoptosis signal-regulatory kinase (ask1), and mapkkk4 are known as mapkkks. downstream targets of p38 mapk are well-known as mitogen and stressactivated protein kinase 1 (msk1), map kinase-interacting kinase 1 (mnk1), and mapk-activated protein kinase 2 and 3 (mapkapk 2 and 3) (freshney et al. 1994 ). these signaling pathway proteins have further downstream targets. for example, mnk1 activates the eukaryotic translation initiation factor 4e (eif4e) (gingras et al. 1999) . mapkapk2 activates heat shock protein 27 (hsp27), camp response element-binding protein (creb), and transcription factor-1 (atf-1) (garrido et al. 2003; tan et al. 1996) . mouse hepatitis virus (mhv) a59 strain induces interleukin-6 (il-6) production via eif4e phosphorylation under p38 mapk activation (banerjee et al. 2002) . inhibitors of p38 mapk inhibit transcription of viral mrna and production of viral protein, indicating that p38 mapk is utilized to promote viral protein synthesis. conversely, p38 mapk enhances transcription of chop (c/ebp homologous protein) encoded by the growth arrest-and dna damage-inducible gene 153 (gadd153) (wang et al. 1996) . the p38 mapk-induced apoptosis via activation of the chop pathway occurs in cells infected with japanese encephalitis virus (jev) (su et al. 2002) . the h5n1 subtype of influenza virus induces tumor necrosis factor alpha (tnf-a) expression via activation of p38 mapk (lee et al. 2005) . the p38 mapk signaling pathway is thought to primarily induce apoptosis in virus-infected cells. as described in hcv infection, p38 mapk is able to promote cell survival. although p38 mapk is activated in many cases of viral infection, viral proteins sometimes negatively regulate p38 mapk. the orf 61 protein of varicella-zoster virus (vzv) is known to repress phosphorylation of p38 mapk for negative regulation of cellular proinflammatory responses (rahaus et al. 2005 ). thus, activation or inactivation of p38 mapk occurs in the pathogenesis of disease caused by viral infection. the p38 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 e6 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. 2004c; yan et al. 2004) . cytopathic effects (cpes), defined as focal cell rounding and dna fragmentation typical of apoptosis, are observed in sars-cov-infected vero e6 cells at 24 h post-infection (h.p.i.) (mizutani et al. 2004c ). activated caspase 3, which has an essential role in apoptosis, was detected at peak levels at 24 h.p.i. on the other hand, the phosphorylation level of p38 mapk reached a maximum at 18 h.p.i. in virus-infected cells. the phosphorylated p38 mapk was active, as shown by using an in vitro kinase assay. the cpe observed in sars-cov-infected cells is slightly inhibited by sb203580, and therefore p38 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 p38 mapk is a promoter of cell death in vero e6 cells infected by sars-cov. however, sb203580 treatment of vero e6 cells indicates that there is no requirement for p38 mapk activation in sars-cov replication. the p38 mapk signaling pathway perhaps has other roles in sars-cov-infected cells. the downstream targets of p38 mapk are phosphorylated in sars-covinfected cells. the level of phosphorylated eif4e is increased in sars-covinfected cells (mizutani et al. 2004c ). however, the activated eif4e does not regulate viral protein synthesis, as demonstrated by the similar kinetics of viral protein accumulation in infected vero e6 cells in the presence and absence of sb203580. both mapkapk-2 and its substrate hsp-27 are phosphorylated in virus-infected cells. hsp-27 is known as an anti-apoptotic protein as it inhibits apoptosome formation (garrido et al. 2003) . creb is also known to mediate a survival signal under various conditions (tan et al. 1996; ginty et al 1994; von knethen et al. 1998) , and creb is also phosphorylated in sars-cov-infected cells. the expression of sars-n protein in transfected cos-1 cells induces phosphorylation of p38 mapk, hsp-27, and creb (surjit et al. 2004) , whereas the viral-n protein expression system of vaccinia virus (dis-n) does not induce phosphorylation of p38 mapk (mizutani et al. 2006d ). activation of the p38 mapk pathway induces actin reorganization in cos-1 cells devoid of growth factors (surjit et al. 2004) . furthermore, the 7a protein of sars-cov induces apoptotic cell death and phosphorylation of p38 mapk in 293 t cells (kopecky-bromberg et al. 2006 ). however, sb203580 does not prevent cell rounding, apoptosis, and chromatin condensation induced by the 7a 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 p38 mapk have the potential to induce an anti-apoptotic environment in sars-cov-infected cells. however, activated p38 mapk in sars-cov-infected cells is thought to be able to promote both cell death and survival. perhaps there are other substrates of p38 mapk that are inducible on cell death of vero e6 cells caused by sars-cov infection, or perhaps there is cross-talk between the p38 mapk signaling pathway and other signaling pathways inducing cell death. in vero e6 cells, signal transducer and activator of transcription (stat) 3 protein is constitutively phosphorylated at tyr-705 and is slightly phosphorylated at ser-727 (mizutani et al. 2004a ). sars-cov infection is able to induce dephosphorylation of stat3 tyr-705 after 18 h.p.i. on the other hand, ser-727-phosphorylated stat3 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 sh2 phosphotyrosine interaction (shuai et al. 1993 (shuai et al. , 1994 schindler et al. 1992a schindler et al. , 1992b . stat3 is known to be activated in response to interleukin-6 (il-6) and il-10, and is thought to act as an anti-apoptotic transcription factor (rajan and mckay 1998; grandis et al. 2000; mora et al. 2002) . tyr-705 phosphorylation of stat is necessary for its activation (shuai et al. 1993 (shuai et al. , 1994 schindler et al. 1992b) , suggesting that sars-cov infection leads to a decrease in stat3 activation. furthermore, stat3 does not act as a transcriptional enhancer in sars-cov-infected vero e6 cells, as shown by the disappearance of tyr-705-phosphorylated stat3 from the nuclear fraction post sars-cov infection. the proteins upstream of stat3 in the signaling pathway are janus kinases (jak1 and 2) and tyk2, which are phosphorylated at low levels in mock-infected vero e6 cells, even after virus infection. the signal transducing adaptor molecule 1 (stam1), which is known to be associated with jak2 and jak3 via the immunoreceptor tyrosine-based activation motif, is upregulated in sars-cov-infected vero e6 cells (leong et al. 2005) . therefore, tyr-705 dephosphorylation of stat3 in virus-infected cells is independent of its upstream kinases, and there may be other signaling pathways regulating stat3 phosphorylation and dephosphorylation. two inhibitors of p38 mapk (sb203580 and sb202190) partially inhibit dephosphorylation of stat3 at tyr-705, indicating that the p38 mapk signaling pathway is upstream of tyr-705 dephosphorylation of stat3 in sars-cov-infected vero e6 cells. inactivation of stat3 via p38 mapk activation may induce cell death in sars-cov-infected cells. however, the kinetics of stat3 after sars-cov infection varies for different cell types. the suppressors of cytokine signaling-3 (socs3) mrna are suppressed in sars-cov-infected caco-2 cells (okabayashi et al. 2006) , leading to continuous activation of stat3. a serine/threonine kinase, p90 ribosomal s6 kinase (rsk), belongs to another signaling pathway, which is regulated by p38 mapk. generally, p90rsk is phosphorylated at thr-573 by erk (gavin and nebreda 1999; smith et al. 1999) , and this phosphorylation induces autophosphorylation at ser-380, and then pdk1 (phosphoinositide-dependent kinase 1) phosphorylates at ser-221 (frã¶din et al. 2000; jensen et al. 1999; richards et al. 1999) . no significant differences are observed in phosphorylation levels of p90rsk at ser-221 and thr-573 in sars-cov-infected vero e6 cells (mizutani et al. 2006a ). however, ser-380 of p90rsk is phosphorylated in virus-infected confluent cells. thus, phosphorylation of p90rsk ser-380 is upregulated without upregulation of thr-573 in sars-covinfected cells. the phosphorylation of ser-380 is decreased in sb203580-treated virus-infected cells, indicating that p38 mapk can induce phosphorylation of ser-380. furthermore, p90rsk phosphorylates creb . in sars-cov-infected vero e6 cells, p38 mapk activation induces phosphorylation of p90rsk ser-380, and then creb is thought to be phosphorylated by activated p90rsk. thus, p90rsk may have anti-apoptotic activity in sars-cov-infected cells. the sars-cov s protein is able to induce phosphorylation of erk1/2 in hek293t cells (liu et al. 2007 ). the s-induced protein kinase c (pkc)/erk signaling pathway promotes nuclear factor-kappa b (nf-kb) binding to the cyclooxygenase-2 (cox-2) promoter. similar results have been reported using the n protein of sars-cov (yan et al. 2006 ). sars-cov s protein expression induces release of interleukin-8 (il-8) via erk and p38 mapk signaling pathways including activator protein 1 (ap-1) in a549 cells ). on the contrary, phosphorylation of erk1/2 is downregulated in n protein-expressing cos-1 cells in the absence of serum (surjit et al. 2004 ). thus, viral proteins can potentially upor downregulate phosphorylation of erk1/2. erk1/2 is observed to be phosphorylated in sars-cov-infected vero e6 cells (mizutani et al. 2004a ). after treatment with mapk/erk kinase 1 and 2 (mek1/2)-specific inhibitor (pd98059), sars-cov-infected vero e6 cells exhibit no significant changes in activated caspase-3 or caspase-7. thus, activation of erk1/2 is not sufficient to prevent cell death by sars-cov infection. furthermore, activation of erk1/2 is not necessary to establish persistent infection of sars-cov in vero e6 cells (mizutani et al. 2005 ). the sars-cov s protein induces creb binding to cox-2 promoter mediated via the phosphatidylinositol 3-kinase (pi3k)/pkc/jnk pathway in hek293t cells (liu et al. 2007 ). expression of the sars-cov n protein induces phosphorylation of jnk in vero e6 cells (mizutani et al. 2006d ) and in cos-1 cells in the absence of serum (surjit et al. 2004 ). 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-1, which is composed of homodimers and heterodimers of fos, jun, creb, and activating transcription factor (atf) subunits, in vero and huh7 cells . the viral accessory proteins, 3a and 7a, phosphorylate jnk1 and jnk3 in hek293t cells (kanzawa et al. 2006) . overall, viral proteins are able to induce phosphorylation of jnk in several cell lines. sars-cov infection induces phosphorylation of jnk in vero e6 cells after at least 12 h.p.i. (mizutani et al. 2004a ). the vero e6 cells begin to show rounding at 24 h.p.i and persistently infected cells are observed after 48 h.p.i (mizutani et al. 2005) . at this time, jnk, akt, and p38 mapk are phosphorylated in virus-infected cells. treatment with an inhibitor of jnk (sp600125), and pi3k (ly294002), inhibits the establishment of persistence, whereas treatment with an inhibitor of mek1/ 2 (pd98059) and p38 mapk (sb203580) does not inhibit persistence of infection (mizutani et al. 2005) . thus, two different signaling pathways of jnk and pi3k/ akt are important for the establishment of persistently infected vero e6 cells (mizutani et al. 2006d . akt, which is also known as protein kinase b (pkb), is phosphorylated at both ser-473 and thr-308 residues via the pi3k signaling pathway upon stimulation by growth factors, insulin, and hormones (toker 2000; brazil and hemmings 2001; scheid and woodgett 2003; welch et al. 1998) . the main role of akt is inhibition of apoptosis via phosphorylation of the forkhead transcription factor (fkhr) family, glycogen synthase kinase-3b (gsk-3b), caspase-9, and bcl-associated death protein (bad) (cardone et al. 1998; cross et al. 1995; datta et al. 1997) . interestingly, gsk-3 regulates phosphorylation of n protein (wu et al. 2009 ). the m protein of sars-cov induces apoptosis in both hek293t cells and transgenic drosophila ). the m protein-induced apoptosis involves mitochondrial release of cytochrome c protein. in sars-cov-infected vero e6 cells, ser-473 of akt is phosphorylated at 8 h.p.i. and maximal phosphorylation is observed at 18 h.p.i. (mizutani et al. 2004b) , after which akt is dephosphorylated. thr-308 phosphorylation has not been detected in vero e6 cells. the phosphorylation of ser-473 of akt by viral infection is inhibited by ly294002, which is an inhibitor of the pi3k 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 e6 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. 2005 (mizutani et al. , 2006d , suggesting that activation of pi3k/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 e6 cells are mainly derived from experiments using confluent cells. when subconfluent vero e6 cells are infected by sars-cov, cell proliferation is inhibited (mizutani et al. 2006c ). 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 e6 cells (liao et al. 2005 ). as described above, the s-and n-induced pkc/erk signaling pathway promotes nf-kb binding to the cox-2 promoter (liu et al. 2007; yan et al. 2006) . sars-cov s and n proteins may cause inflammation of the lungs by activating cox-2 gene expression. the 3a and 7a viral accessory proteins enhance nf-kb mediated transcription in hek293t cells (kanzawa et al. 2006) . in contrast, the n protein inhibits interferon production in 293 t cells via inhibition of nf-kb (kopecky-bromberg et al. 2007 ). the m protein also suppresses nf-kb activity (fang et al. 2007) . growth arrest and apoptosis via caspase-3 and caspase-9 activities are induced in sars-cov 3c-like protease (3cl pro )-expressing human promonocyte hl-cz cell line (lin et al. 2006) . the sars-cov 3cl pro may increase activation of nf-kb and upregulate cytochrome c oxidase and downregulate hsp-70, inducing mitochondrial-mediated apoptosis (lai et al. 2007 ). viral papain-like protease (plp) regulates antagonism of irf3 and nf-kb signaling pathways (frieman et al. 2009 ). the 3a protein of sars-cov has the potential to inhibit cell cycle progression at the g 1 phase in hek293, cos-7, and vero cells (yuan et al. 2005 (yuan et al. , 2007 . the c-terminal region of the 3a protein, which includes a potential atpase motif, is essential to inhibit the cell cycle. the 3a protein expression reduces cyclin d3 level and inhibits retinoblastoma (rb) phosphorylation. the p53 phosphorylation is increased by 3a expression. the 7a protein expression also blocks cell cycle progression at the g 0 /g 1 phase in hek293, cos-7, and vero cells by mechanisms similar to those of the 3a protein (yuan et al. 2006) . the n protein is a substrate of cyclin-dependent kinase (cdk) as well as gsk, mapk, and casein kinase ii (surjit et al. 2005) . the n protein directly binds to cyclin d and inhibits activity of the cyclin d-cdk4 complex. the n protein also inhibits cdk2 activity by direct binding to the cdk2-cyclin complex, resulting in blocking the s phase progression in cos-7 and huh7 cells (surjit et al. 2006) . 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 e6 cells, via dephosphorylation of stat3 by p38 mapk activation, and inactivation of akt, as previously described. recent study suggest that sars-cov triggers apoptosis via protein kinase r (pkr) (krã¤hling et al. 2009 ). 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 3cl pro expression in hl-cz cells induces apoptosis via caspase-3 and caspase-9 (lin et al. 2006) . furthermore, 3cl pro expression in hl-cz cells upregulates proteins located in the mitochondria, but downregulates hsp-70, which antagonizes apoptosis-inducing factor (lai et al. 2007 ). the sars-cov 8a protein, localized in the mitochondria of infected cells, increases mitochondrial transmembrane potential, reactive oxygen species production, and caspase-3 activation, resulting in inducing apoptosis in vero, hek293, and huh7 cells ). orf 6 induces apoptosis via caspase-3 mediated, er stress and jnkdependent pathways (ye et al. 2008 ). sars-cov n protein modulates the tgf-b signaling pathway to block apoptosis of sars-cov-infected host cells (zhao et al. 2008) . 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. 2004) in cos-1 cells, but not in hep-g2 and huh-7 cells ). the sars-cov s protein and its c-terminal domain (s2) induce apoptosis in vero e6 cells, but the s1, e, m, and n proteins are not able to induce apoptosis in vero e6 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 hek293t cells and transgenic drosophila [85]. overexpression of sars-cov 3a protein in vero e6 cells induces apoptosis, mediated through a caspase-8-dependent pathway or p38 mapk waye et al. 2005; padhan et al. 2008) . the 3a protein expression in drosophila induces apoptosis, which could be modulated by cellular cytochrome c levels and caspase activity . the sars-cov 3b protein induces both necrosis and apoptosis in vero e6 cells (khan et al. 2006) . the sars-cov 7a protein interacts with pro-survival proteins, basal cell lymphoma-extra large (bcl-xl), b cell lymphoma 2 (bcl-2), bcl-w, a1, and myeloid cell leukaemia sequence 1 (mcl-1), at the endoplasmic reticulum and the mitochondria, resulting in triggering apoptosis in hek293t and vero e6 cells (tan et al. 2007 ). interestingly, the 7a protein does not interact with the pro-apoptotic members, bcl-2 associated x protein (bax), bcl-2 homologous killer (bak), bad, and bcl-2 interacting domain (bid). however, a mutant virus without the 7a/7b gene is able to induce extensive cpes in the vero cell line (yount et al. 2005) , suggesting that the 7a 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-2 in cos-1 cells (surjit et al. 2004 ). 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-2 in sars-covinfected vero cells (bordi et al. 2006 ). in the virus-infected vero cells, downregulation of bcl-2 and upregulation of bax are observed (ren et al. 2005) . bcl-xl activation plays important roles in establishing persistent infection of sars-cov (mizutani et al. 2006b) . the n protein upregulates the bcl-xl protein level (mizutani et al. 2006d ). 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-p38 indicated that augmented p38 mapk phosphorylation of cd14 monocytes was associated with suppressed p38 mapk phosphorylation of cd8 lymphocytes, suggesting that altered leukocyte p38 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-1 (svcam-1) levels and soluble fas ligand (sfasl) levels, as determined using elisa (enzyme-linked immunosorbent assay). sars patients also have intracellular activated caspase-3 fragment levels, as measured using flow cytometry (peiris et al. 2003b) . liver impairment commonly occurs amongst patients with sars, indicating that sars-cov may be localized in the liver (chau et al. 2004 ). 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. 2003) . myd88-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. 2008) . 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. 19.2) . the balance of activities of signaling pathways is important for determination of cell death or 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cells sars coronavirus 7a protein blocks cell cycle progression at g0/g1 phase via the cyclin d3/prb pathway g1 phase cell cycle arrest induced by sars-cov 3a protein via the cyclin d3/prb pathway sars-cov nucleocapsid protein induced apoptosis of cos-1 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 smad3 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-002935-jq1xumrh 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: 2018-03-22 journal: plos one doi: 10.1371/journal.pone.0194880 sha: doc_id: 2935 cord_uid: jq1xumrh 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 50% and/ or 90% inhibition (ec(50), ec(90)) 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(50). 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 [1] [2] [3] [4] . ebov is a biosafety level-4 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 [5] [6] [7] [8] . 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 2013-2016 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 [9, 10] . 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 [11] [12] [13] [14] . 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-4 viral pathogen. classical methods evaluate drug efficacy include the reduction of virus yield [15] or a decrease in viral rna transcription as determined by real-time polymerase chain reaction (pcr) [16] [17] [18] . 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 [19] . 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 [20] [21] [22] [23] . 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 vp40-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 [24] [25] [26] [27] . 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 50 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 e6 (african green monkey kidney; atcc 1586) cells were obtained from the american type culture collection (manassas, va). vero c1008 (e6) cells (african green monkey kidney, working cell bank nr-596) were obtained through bei resources (national institute of allergy and infectious diseases [niaid] , national institutes of health [nih], manassas, va). huh 7 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 e6 and huh 7 cells culture were grown to 90% confluency in a t-175 (fisher scientific) or triple layer tissue culture flask (nunc) containing dulbecco's modification of eagle medium (dmem) (gibco) supplemented with 10% 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 4-22, at which point a new culture was introduced and the previous passage series was ended. additionally, cell cultures were required to be a least 85% 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 [28, 29] . briefly, pbmcs were isolated from human whole blood by density-gradient centrifugation over histopaque (1.077 g/ml, sigma-aldrich, st. louis. mo). monocytes were purified using human cd14-specific microbeads (miltenyi biotec, san diego, ca, 130-050-201) following manufacturer's instructions. cd14 + monocytes were differentiated into mdms by culturing for 6-7 days with recombinant human macrophage colony-stimulating factor (bio-techne, minneapolis, mn, 216-mc-005) and conditioned medium from kpb-m15 cells (kind gift from dr. atsunobu hiraoka, scgf research laboratory, kyoto, jp). media were replaced every 2-3 days during the incubation for a total of 6-7 days. the cells were harvested and plated on desired 96-well plates 1 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 89778-27-8; t7204-5mg). the makona 05 isolate of ebov (h. sapiens-tc/gin/14/wpg-c05) (ebov/mak, genbank accession no. kp096420), 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 0.01 in vero c1008 cells (bei resources, manassas, va, catalog nr-596). when the cytopathic effect was visible at day 5-7 after infection (51-75% of monolayer showing cpe), cell culture supernatants were harvested and clarified by centrifugation. the ebov/mak titer was determined by plaque assay in vero e6 cells. virus titers were measured using 10-fold serial dilutions of culture supernatant in triplicate infections of vero e6 cell monolayers in 6-well plates. after incubation at 37˚c for 1 h (plates were rocked every 15 minutes), 2 ml of medium containing 2x mem (gibco), 1.25% avicel (fmc biopolymer), and 1x antibiotic-antimycotic (gibco) were added to each well (2ml/well). after 7 days post-incubation, virus plaques were stained with 0.2% crystal violet (ricca chemical) in 10% 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-4 (bsl-4) conditions. vero e6 and huh 7 cells were seeded overnight at 3 to 4 × 10 4 cells per well and mdm cells were plated at 1 × 10 5 cells per well in 100 μl of dulbecco's modified eagles's medium with 10% fetal bovine serum in black opaque (thermo fisher scientific, waltham, ma, corning 3916, 07-200-627) or clear bottom 96-well greiner microplates (greiner bio-one, monroe, nc, 655948). ebov/mak isolate was diluted in culture media to the specified mois (the titers used to determine moi hereby were generated on vero e6 cells) in 96-well plates. the cells were then infected by transferring 50 μl of ebov/mak isolate from the virus dilution plates to cell plates using the 96-well liquidator (rainin instrument, oakland, ca). the cells were incubated at 37˚c and 5% co 2 for the indicated periods of time. the plates were fixed by adding 200 μl of 20% neutral-buffered formalin (final concentration 10%) at 24, 48, 72 or 96 h postinoculation (hpi). after fixing for 24 h, the plates were transferred to a bsl-2 lab for antibody staining as described previously [28] . briefly, ebov was detected by exposure of the infected, fixed, and permeabilized cells to a monoclonal mouse antibody specific to the ebov vp40 matrix protein (b-md04-bd07-ae11, made by us army medical research institute of infectious diseases, frederick md under centers for disease control and prevention contract) [30] , followed by staining with an alexafluor 1 594 goat anti-mouse igg (heavy + light chains) antibody (life technologies, carlsbad, ca) at 37˚c for 1 h. fluorescence was quantified using a tecan plate reader (infinite 1 m1000, tecan us, morrisville, nc) or an high-content imaging (hci) system (operetta, perkinelmer, waltham, ma). hci images were collected at 20x magnification using 1 to 9 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 33342 dye (molecular probes) at 37˚c for 2 h. columbus 2.4.2 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 vp40 antibody followed by staining with the horseradish peroxidase (hrp)-conjugated goat anti-mouse secondary antibody (seracare, milford, ma, cat. #074-1802). chemiluminescence was quantified using pico chemiluminescent substrate (thermo fisher scientific inc., rockford, il) and a plate reader (infinite 1 m1000 tecan). vero e6, huh 7 and mdm cells were seeded as described above in 100 μl media overnight at 37˚c with 5% co 2 . compounds in dimethyl sulfoxide were prediluted to reduce dimethyl sulfoxide concentration to 0.05% or lower. compounds were prediluted in dilution blocks before performing a final 1:4 dilution by transferring 50 μl of each compound to cell plates containing 150 μl of cell culture media. this dilution achieved a desired compound concentration in 200 μl of cell culture media. the process of performing the drug screen assay is shown in fig 1. three plates were set up per experiment, two plates (clear bottom 96-well greiner microplates) for detecting inhibition of ebov, and one mock plate (black opaque plate) for determining drug cytotoxicity. after 1 h of predilution and transport to the bsl4 laboratory, 50 μl of virus (or mock control) at the desired moi was added to cells. at 48, 72 or 96 hpi, assay plates were fixed at final concentration of 10% nbf for 24 h before transferring to a bsl-2 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 48 or 72 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 1 m1000 tecan plate reader (fig 1) . non-linear regression analysis and curve fitting parameter were performed to calculate ec 50 s, ec 90 s and 50% cytotoxic concentration (cc 50 s) (graphpad software, la jolla ca) [12] 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 [31, 32] , and ec 50 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-2. immunostaining was performed with a ebov-specific antibody against vp40 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 33342). in parallel, cytotoxicity assays (celltiter glo) with mock infected cells are performed at bsl-2. luminescence is read on the infinite 1 m1000 tecan plate reader. data are analyzed using graphpad prism and/or columbus software (operetta). https://doi.org/10.1371/journal.pone.0194880.g001 factors that influence ebola antiviral activities in cell-based assays plos one | https://doi.org/10.1371/journal.pone.0194880 march 22, 2018 represented by z' factor which is defined as equation z' = [1-((3 ã sd pos )+(3 ã sd neg ))/(imean pos -imean neg )]. in our assay, the z' criteria is as follows: z' = 0.5-1.0 corresponds to an excellentassay; z' = 0-0.5 corresponds to a suboptimal assay; z' <0 corresponds to a unsuccessful assay [32] . the susceptibility to ebov infection was evaluated in multiple cell types in cell-based assays measuring anti-ebov activity. vero e6, huh 7 and mdm cells were infected with ebov/ mak isolate at 8 different mois, and the assay was terminated after 24, 48, 72 or 96 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 vp40 positive cells. the growth of ebov/mak isolate in three cell types was compared over time. in vero e6 cells, ebov spread slightly slower, and the number of positive cells were overall lower compared to huh 7 cells (fig 2a-2d ). mdms were the most susceptible to ebov among the cell types. at 24 hpi, mdms already exhibited a typical dose response relative to virus input, while only minimal ebov replication was observed in vero e6 and huh 7 cells at 24 hpi at all mois tested. virus spread effectively at 48 hpi with higher (1 to 3.3) moi of vero e6, huh 7 cells and most of mois in mdms (fig 2a-2f ). the infection in mdms was saturated at 72 hpi at almost all mois (fig 2e and 2f ). 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 96 hpi time point and at higher virus input (mois of 3.3) (fig 2b, 2d , 2f and 2g). 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 2g) . 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 e6 and huh 7 cells (tables 1 and 2 ). the same experimental plates from fig 2a-2d 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 10 to 80% and the s/n ratio at 4 to 10. at lower s/n ratios (<4) or lower percentage of infected cells (<10%) distinguishing activity of a drug from non-activity becomes increasingly difficult. on the other hand, at higher s/n ratios (>10), when cell infection rate trends towards 80-90% 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 2 and tables 1 and 2 , two optimized conditions for the fluorescence assays were selected. an moi of 1 with 48 hpi as endpoint and an moi of 0.1 with an endpoint of 72 hpi produced a robust signal for all 3 cell types, vero e6, huh 7 and mdms (fig 2a-2f , tables 1 and 2). vero e6 cells were infected with ebov at around 17.1%, and mdms and huh 7 cells usually had a stronger signal with around 80% or higher of ebov-positive cells (fig 2c-2f ). the two conditions had a similar signal or ebov infection during the assay development phase, vero e6 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 e6 and huh7 cells were analyzed at different cell passage numbers by determining percentage of ebov-positive cells (fig 3a and 3b ). both cell types were infected with a moi of 1 for 48 h. the viral spread of huh 7 cells were consistent over cell culture passages 6-30, indicating that the passage number does not impact ebov spread c the percentage of ebov-positive cells was determined with a vp40/alexa-594 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 3 infected wells) to the noise (mean from 3 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 7 cells in the range tested. in contrast, ebov spread on vero e6 cells varied considerably between cell passages 6-28 with peak infection (replication efficacy) at cell culture passages 12-14 ( fig 3a) and equally lower for early or late passages. significant differences were observed at passages 8, 10, 12, 14, and 16 when compared to the passage 6 or 28 using an ordinary one-way anova following dunnett's multiple comparison in graphpad prism 7.0. occasionally, exceptions to this trend were observed (fig 4) . the infectivity of ebov in vero e6 and huh 7 cells were compared at an early (p6) and a late passage number (p28) at a range of mois (0.03-2.0) for 48 h. in this case, vero e6 cells tested at the later passage showed a lower infection rate (<40%) as expected, while the early passage demonstrated an unusually high infection rate (up to 80%). infectivity in huh 7 cells remained consistent regardless of passage number, vero e6 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 [33] , under different parameters using fluorescence as the read out. huh 7 cells were infected with a constant moi of 1 and treated with toremifene citrate for 48, 72, or 96 h (fig 5a) . later time points resulted in a decrease in activity with the ec 50 at 96 hpi 3.3-fold higher than at 48 hpi. when the time point remained constant at 72 hpi the activity of toremifene citrate increased as the virus input decreased (mois of 0.1, 0.3, 1, or 3) (fig 5b) . the ec 50 at a moi of 0.1 was 6.9-fold lower than at a moi of 3. in addition to time and virus input, the cell type used in the assay can result in differences in the calculated ec 50 . anti-ebov activity of toremifene citrate was measured using variable assay endpoints (48, 72 and 96 hpi), mois (0.001, 0.01, 0.1, and 1), and cell lines (vero e6 and huh 7 cells, fig 6) . ec 50 values for toremifene citrate increased with exposure time and virus input in both cell types. overall, activity in vero e6 cells was higher with maximum activity (ec 50 = 0.15 μm) at 96 hpi and an moi of 0.01. in huh 7 cells, maximum activity (ec 50 = 0.28 μm) was detected at 96 hpi and an moi of 0.001. ec 50 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 [34, 35] . 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 7 cells with a range of mois (0.1, 0.3, 1, and 3 ) at three different time points (24, 48 and 72 hpi) . assay parameters, signal-to-noise (s/n), and z' factor, were compared between the assays (table 3) . at the earliest time point (24 hpi), the celia had higher sensitivity than the fluorescent detection assay as the z' were in the acceptable range (>0.2) even at the lowest moi of 0.1. in contrast, the fluorescent assay required higher mois for reliable data sets (z'>0.2). at the 48 hpi time point, both detection methods generated high quality data sets with the celia providing improved z' factors (>0.8). at 72 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 50 s and ec 90 s factors that influence ebola antiviral activities in cell-based assays were determined on those data sets with acceptable z' (>0.2) and overall there was good correlation between the different detection methods (table 4 ). all detection assays demonstrated that higher moi and longer times of exposure led to an increase of the ec 50 values for toremifene citrate. in contrast, the ec 90 values showed considerably lower fluctuations under the different conditions tested. the established celia was used to compare anti-ebov activity of toremifene citrate in 3 different cell types, vero e6, huh 7 and mdms using an moi of 0.5 and a time point of 48 h (fig 7) . huh 7 cells and mdms had s/n ratios in the range of 1000s with z' factors generally above 0.5 (fig 7a) . the s/n for vero e6 cells ranged from >100 to over 1000, leading to more variability as reflected in the wider range of z' factors (0.3-0.9) (fig 7a) . the activity (ec 50 ) of toremifene was compared, and considerable differences were detected for the 3 cell types ( fig 7b) . the efficacy of toremifene citrate was 3-fold higher in huh 7 cells than in mdms, and 2-fold higher than in vero e6 cells (fig 7b) . 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 [36] . the vero e6 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 e6 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 7 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 [37] . therefore, a drug that is highly effective at inhibiting ebov infection in mdms may better translate to in vivo potency than vero e6 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 24 hpi at an of moi 0.1. in contrast, ebov grew slower in vero e6 and huh 7 cells. the optimal conditions for evaluation of drug efficacy using the fluorescent assay were at an moi of 0.1 for the 72 h time point or at an moi of 1 for the 48 h time point for all 3 cell types to ensure a strong virus signal and avoid destruction of cell layer (fig 2g) . however, an moi of 1 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 e6 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 [38, 39] , we found that at lower or higher passages the vero e6 cells can show a decreased percentage of ebov-positive cells. huh 7 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 1 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 8 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 24 hpi with the lowest virus input detected at an moi of 0.1. an ec 50 could also be determined at this time point. at 48 or 72 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 [12, 33] . this is an fda-approved drug for treating breast cancer [12] . mechanism of action studies showed that toremifene citrate affects ebov virus entry at the stage of virus-fusion with the endosomal membrane [40] . 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 50 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 90 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 [41] showing that activity of brincidofovir was dependent on media conditions with low serum (< 5%) and 48 h pretreatment before adding virus. in contrast, the serum concentration had no effect on the activity of toremifene citrate, and pretreating cells for 48 h (instead of 1 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 50 (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 50 of a drug can range over more than a log based on testing conditions (e.g, moi, time of endpoint, serum). johanson et. al. [12] reported ec 50 s of 1.73 and 0.97 μm for toremifene with two different ebov strains ebov/kik and ebov/may, respectively, at 48 hpi with 0.4 moi by celia using the 13c6 antibody. these data are comparable to the ec 50 of 1.10 ± 0.71 μm for ebov/mak determined in our assay under similar conditions (table 4 ; moi 0.3/ 48 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 1 clinical trial of apical membrane antigen 1: an asexual blood-stage vaccine for plasmodium falciparum malaria the soviet union's anti-agricultural biological weapons hemorrhagic fever viruses as biological weapons: medical and public health management transmission of ebola virus (zaire strain) to uninfected control monkeys in a biocontainment laboratory lethal experimental infections of rhesus monkeys by aerosolized ebola virus drug repositioning: identifying and developing new uses for existing drugs treatment of ebola virus disease productive replication of ebola virus is regulated by the c-abl1 tyrosine kinase fda-approved selective estrogen receptor modulators inhibit ebola virus infection a systematic screen of fda-approved drugs for inhibitors of biological threat agents screening of fda-approved drugs for treatment of emerging pathogens plaque assay for ebola virus rapid detection and quantification of rna of ebola and marburg viruses, lassa virus, crimean-congo hemorrhagic fever virus, rift valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-pcr identification of bisindolylmaleimides and indolocarbazoles as inhibitors of hcv replication by tube-capture-rt-pcr high-throughput molecular detection of hemorrhagic fever virus threats with applications for outbreak settings generation of egfp expressing recombinant zaire ebolavirus for analysis of early pathogenesis events and high-throughput antiviral drug screening novel small molecule entry inhibitors of ebola virus identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay identification of an antioxidant small-molecule with broad-spectrum antiviral activity identification of a small-molecule entry inhibitor for filoviruses toremifene for breast cancer: a review of 20 years of data phase ii clinical trial of high-dose toremifene as primary hormone therapy in aromatase inhibitor-resistant breast cancer toremifene citrate (fareston) evaluation of the activity of lamivudine and zidovudine against ebola virus characterization of yellow fever virus infection of human and non-human primate antigen presenting cells and their interaction with cd4+ t cells pathology of experimental ebola-zaire (mayinga) virus infection transmitted to guinea pigs by oral, conjunctival and tonsillar routes high-throughput screening assays for the identification of chemical probes a simple statistical parameter for use in evaluation and validation of high throughput screening assays a screen of approved drugs and molecular probes identifies therapeutics with anti-ebola virus activity determination of residual enrofloxacin in food samples by a sensitive method of chemiluminescence enzyme immunoassay chemiluminescence enzyme immunoassay for the determination of sulfamethoxydiazine development of high-content imaging assays for lethal viral pathogens drug targets in infections with ebola and marburg viruses use of multiple assay endpoints to investigate the effects of incubation time, dose of 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-m15 cells. we thank dr. gary kobinger (public health agency of canada, winnipeg, ca) for the makona isolate of ebola virus (genbank accession no. kp096420). we thank dr. hideki ebihara (niaid, rocky mountain laboratories, hamilton, mt) for huh 7 (human hepatocellular carcinoma) cells. we thank dr. sheli radoshitzky (usamriid, frederick md) for her gift of vp40 ebov vp40 matrix protein (b-md04-bd07-ae11. in key: cord-272729-nbgdmavr authors: kim, youngnam; lee, changhee title: ribavirin efficiently suppresses porcine nidovirus replication date: 2012-10-27 journal: virus res doi: 10.1016/j.virusres.2012.10.018 sha: doc_id: 272729 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, 1997; mayo, 2002; spaan et al., 2005) . 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, 1a and 1b, comprising the 5 two-thirds of the viral genome encoding non-structural proteins (nsps) and the remaining orfs located in the 3 terminal region coding for structural proteins (lai et al., 2007; snijder and spaan, 2007) . the initial translation from orf1a and orf1b yields the 1a and lab replicase polyproteins, respectively, which are then proteolytically processed into functional nsps including the viral rna-dependent rna polymerase (rdrp) (bautista et al., 2002; van aken et al., 2006; ziebuhr et al., 2000) . the rdrp-containing replication complex mediates genomic rna replication and subgenomic (sg) mrna transcription, eventually generating a nested set of 3 -coterminal sg mrnas that are individually translated to structural proteins (lai et al., 2007; snijder and spaan, 2007) . 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, 1997) . 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., 1994; christopher-hennings et al., 1995; duan et al., 1997; wills et al., 2003) . 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., 2000; feng et al., 2001; harms et al., 2001; wills et al., 2000) . 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, 1980) . 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., 2005; pensaert and yeo, 2006) . ribavirin (1-␤-d-ribofuranosyl-1,2,4-triazole-3-carboxamide, also known as virazole) is a synthetic guanosine analog that exhibits broad-spectrum antiviral activity in vitro (sidwell et al., 1972) . 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, 2007; cooper et al., 2003; day et al., 2005; mccormick et al., 1986; lanford et al., 2001; rockx et al., 2010; severson et al., 2003) . most notably, ribavirin is used in combination with interferon-␣ for treatment of chronic hepatitis c virus (hcv) infections (cummings et al., 2001; davis et al., 1998) . 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-pcd163 cells were cultured in rpmi 1640 medium (invitrogen, carlsbad, ca) supplemented with 10% fetal bovine serum (fbs, invitrogen), antibiotic-antimycotic solutions (100×; invitrogen), 10 mm hepes (invitrogen), 1 mm sodium pyruvate (invitrogen), and nonessential amino acids (100×; invitrogen) in the presence of 50 g/ml zeocin (invitrogen). vero cells were cultured in alpha minimum essential medium (␣-mem, invitrogen) with 10% fbs and antibiotic-antimycotic solutions. the cells were maintained at 37 • c in a humidified 5% co 2 incubator. prrsv strain vr-2332 was propagated in pam-pcd163 cells as described previously . pedv strain sm98-1 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, 1988) . 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; sdow17) 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-pcd163 and vero cells were analyzed by a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (mtt) assay (sigma) detecting cell viability. briefly, pam-pcd163 and vero cells were grown at 1 × 10 4 cells/well in a 96-well tissue culture plate with ribavirin or mpa treatment for 24 h. after one day of incubation, 50 l of mtt solution (1.1 mg/ml) was added to each well and the samples were incubated for an additional 4 h. the supernatant was then removed from each well, after which 150 l of dmso was added to dissolve the color formazan crystal produced from the mtt. the absorbance of the solution was measured at 540 nm using an enzyme-linked immunosorbent assay plate reader. all mtt assays were performed in triplicate. pam-pcd163 and vero cells grown on microscope coverslips placed in 6-well tissue culture plates were pretreated with ribavirin or mpa for 1 h and mock infected or infected with prrsv and pedv at a multiplicity of infection (moi) of 01, respectively. the virusinfected cells were further grown in the presence of ribavirin until 48 hpi, fixed with 4% paraformaldehyde for 10 min at room temperature (rt) and permeabilized with 0.2% triton x-100 in pbs at rt for 10 min. the cells were blocked with 1% bovine serum albumin (bsa) in pbs for 30 min at rt and then incubated with n-specific mab 7 for 2 h. after being washed five times in pbs, the cells were incubated for 1 h at rt with a goat anti-mouse secondary antibody conjugated with alexa fluor 488 (molecular probes, carlsbad, ca), followed by counterstaining with 4 ,6-diamidino-2-phenylindole (dapi; sigma). the coverslips were mounted on microscope glass slides in mounting buffer (60% glycerol and 0.1% sodium azide in pbs) and cell staining was visualized using a fluorescent leica dm il led microscope (leica, wetzlar, germany). pam-pcd163 and vero cells were grown in 6-well tissue culture plates for 1 day and were mock infected or infected with prrsv or pedv at an moi of 0.1. at the indicated times, cells were harvested in 50 l of lysis buffer (0.5% tritonx-100, 60 mm ␤glycerophosphate, 15 mm -nitro phenyl phosphate, 25 mm mops, 15 mm, mgcl 2 , 80 mm nacl, 15 mm egta [ph 7.4], 1 mm sodium orthovanadate, 1 g/ml e64, 2 g/ml aprotinin, 1 g/ml leupeptin, and 1 mm pmsf) and sonicated on ice five times for 1 s each. homogenates were lysed for 30 min on ice, and clarified by centrifugation at 15,800 × g (eppendorf centrifuge 5415r, hamburg, germany) for 30 min at 4 • c. the protein concentrations of the cell lysates were determined by a bca protein assay (pierce, rockford, il). the cell lysates were mixed with 4× nupage sample buffer (invitrogen) and boiled at 70 • c for 10 min. the proteins were separated on nupage 4-12% gradient bis-tris gel (invitrogen) under reducing conditions, and electrotransferred onto immunobilon-p (millipore, billerica, ma). the membranes were subsequently blocked with 3% powdered skim milk (bd biosciences, belford, ma) in tbs (10 mm tris-hcl [ph 8.0], 150 mm nacl) with 0.05% tween-20 (tbst) at 4 • c for 2 h, and reacted at 4 • 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 1:5000 for 2 h at 4 • 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-pcd163 and vero cells were infected with prrsv or pedv, respectively, at an moi of 0.1 as described above. at −1, 0, 1, 2, 4, 6, 8, 10, 12, or 24 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 48 hpi by fixing the monolayers with 4% paraformaldehyde for 10 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., 2007) . briefly, pam-pcd163 and vero cells grown in 6-well culture plates were infected with prrsv and pedv, respectively, at an moi of 0.1 at 4 • c for 1 h, respectively. unbound viruses were then washed with pbs and the cells were either incubated at 4 • c or 37 • c in the presence ribavirin for 1 h, followed by treatment with protease k (0.5 mg/ml) at 4 • c for 45 min to remove the bound but uninternalized virus particles. the prrsv-infected cells were then serially diluted in rpmi and added onto fresh pam-pcd163 cell monolayers in 96-well tissue culture plates. at 48 h post-incubation, internalized viruses were titrated through ifa as described above and the 50% tissue culture infectious dose (tcid 50 ) was calculated. for pedv, the serially diluted infected cells were added onto uninfected vero cells and after 48 h, internalized viruses were titrated using a plaque assay as described previously , and the plaques were counted after 1% crystal violet staining. pam-pcd163 and vero cells were incubated with ribavirin for 1 h prior to infection and then inoculated with prrsv or pedv at an moi of 1 for 1 h at 37 • c. the virus inoculum was subsequently removed and the infected cells were maintained in fresh medium containing ribavirin for 48 h. total rna was extracted from lysates of the infected cells at 48 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 1) as described previously (sagong and lee, 2011) . the rna levels of viral genes were normalized to that of mrna for the ␤-actin or glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene and relative quantities (rq) of mrna accumulation were evaluated using the 2 − 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-pcd163 and vero cells were prrsv or pedv infected with treatment of ribavirin as described above. the culture supernatant was collected at different time points (6, 12, 24, 36, and 48 hpi) and stored at −80 • c. the titer of prrsv was measured by limiting dilution on pam-pcd163 cells through ifa as described above and then quantified as the tcid 50 per ml. the pedv titer was determined by a plaque assay using vero cells and quantified as plaque-forming units (pfu) per ml. pam-pcd163 and vero cells were preincubated with 100 m guanosine for 6 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 48 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 48 hpi, total rna was extracted by trizol, and rt-pcr was performed to amplify the region encoding the orf5 to orf7 genes of prrsv or the n gene of pedv using gene-specific primer sets (table 1) . 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 20 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 0.05 were considered statistically significant. virus-specific cpes were observed daily and were photographed at 48 hpi using an inverted microscope at a magnification of 100× (first panels). for immunostaining, infected cells were fixed at 48 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 200× magnification. viral productions in the presence of ribavirin were measured by quantifying the number of cells expressing n proteins through ifa. five fields at 200× 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 = 0.001-0.05; † p < 0.001. 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-pcd163 and vero cells (data not shown). pam-pcd163 and vero cells were pretreated with ribavirin at concentrations of 10-50 m or 10-200 m, respectively, or with dw as a vehicle control for 1 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 48 hpi (fig. 1) . in vehicle-treated control cells, visible cpe appeared at 24 hpi and became predominant by 48 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. 1a and b, ribavirin significantly decreased virus-induced cpe and viral gene expression of prrsv and pedv at concentrations of ∼10 m and ∼50 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 ∼80% inhibition in response to 50 m and 200 m for prrsv and pedv, respectively ( fig. 1a and b) . moreover, the effective doses for inhibiting 50% (ed 50 ) of the replication of prrsv and pedv were determined to be about 21 m and 73 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-pcd163 and vero cells were treated independently with ribavirin at various time points post-infection. at 48 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. 2) . treatment of cells with 50 m ribavirin for up to 4 hpi resulted in more than 80-90% decrease in prrsv production when compared to the untreated control. addition of ribavirin between 6 and 12 hpi led to 75-38% inhibition of prrsv infectivity. similarly, treatment with 200 m ribavirin at −1, 0, and 1 hpi was found to lead to 83-61% suppression of pedv production, while its treatment at 2-12 hpi resulted in 49-28% reduction in pedv infectivity. in contrast, no significant impairment of porcine nidovirus propagation was observed when ribavirin was added at 24 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 48 hpi and viral titers were measured. as fig. 3a shows, the presence of ribavirin reduced the release of viral progeny in a dose-dependent manner. the peak viral titer was determined to be 10 6 tcid 50 /ml and 10 6.34 pfu/ml in the vehicle-treated control for prrsv and pedv, respectively. however, the addition of 50 m or 200 m ribavirin decreased titers of prrsv and pedv to 10 3.53 tcid 50 /ml and 10 3.52 pfu/ml, respectively (almost 3 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. 3b) . 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., 2007) . pam-pcd163 and vero cells were inoculated with prrsv and pedv, respectively, at 4 • c for 1 h to allow virus attachment and further maintained either at 4 • c or 37 • 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-pcd163 and vero cell monolayers and virus titers were measured 2 days later through ifa or by plaque assay. as shown in fig. 3c , the titers of prrsv and pedv were virtually the same in cells treated with ribavirin or without ribavirin at 37 • c and determined to be 10 1.7 -10 2.2 tcid 50 /ml and 2.2 × 10 2 -3.2 × 10 2 pfu/ml, respectively, indicating no difference between those cells. in contrast, only minimal viral productions of about 10 1.3 tcid 50 /ml (prrsv) and 4.5 × 10 1 pfu/ml (pedv) were observed in cells maintained at 4 • 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-pcd163 and vero cells were treated with ribavirin for 1 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 48 hpi by western blot analysis. the production of both proteins productions was drastically diminished during ribavirin treatment (fig. 4) . 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 90% inhibition at the concentration of 50 m and 200 m, respectively (fig. 4) . 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. 5a , ribavirin exhibited a maximal 70% and 80% 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 4 • c or 37 • 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 2 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 = 0.001-0.05; † p < 0.001. mrna at a concentration of 50 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 200 m ribavirin were only about 13% and 11% of the untreated level (fig. 5b) . 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, 2006) . 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 100 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 29% and 17% at 10 m and 50 m, respectively, whereas supplementation of guanosine to ribavirin inhibited virus production to 40% and 26% at the same concentrations (fig. 6a, left panel) . likewise, while pedv production was declined to 71%, 37%, and 12% in the presence of ribavirin alone at 50 m, 100 m, and 200 m, respectively, it decreased to 100%, 48%, and 31% at the same concentrations when guanosine was added to the ribavirin treatment (fig. 6a, 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. 6b , mpa efficiently reduced porcine nidovirus propagation fig. 4 . inhibition of viral protein translation by ribavirin. ribavirin-treated pam-pcd163 and vero cells were mock-infected or infected with prrsv (a) or pedv (b) for 1 h and were further cultivated in the presence or absence of ribavirin. at 48 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 = 0.001-0.05; † p < 0.001. inhibition of viral rna transcription by ribavirin. pam-pcd163 and vero cells pretreated with ribavirin were mock-infected or infected with prrsv (a) or pedv (b) for 1 h and were incubated in the presence of ribavirin. total cellular rna was extracted at 48 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 = 0.001-0.05; † p < 0.001. at concentrations greater than 0.5 m and 0.1 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 20 recombinant sequences of the orf5 to orf7-coding region corresponding to nucleotide numbers 13,559-15,090 (prrsv) and the n gene region corresponding to nucleotide numbers 26,335-27,660 (pedv) from each virus grown in the presence of ribavirin. as controls, 20 colonies were also obtained individually from untreated prrsv-and pedv-infected a the prrsv orf5-orf7 coding region (1532 bp) and the pedv n (1326 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, 20 clones per group were selected and analyzed the number (rate) of mutations. b a total of 30,640 nt and 26,520 nt for prrsv and pedv were sequenced and a mutation frequency was calculated per 1000 nt, respectively. cells. in the ribavirin-treated groups, point mutations occurred in 17 (85%) plasmids for both prrsv and pedv, which corresponded to a mutation frequency of 1.49 and 1.66 per 1000 nt, respectively. interestingly, mutations were also found in 17 (85%) plasmids in the untreated groups for prrsv and pedv, which were calculated as a mutation frequency of 1.69 and 1.24 per 1000 nt, respectively (table 2) . 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, 2002; lai et al., 2007; snijder and spaan, 2007) . 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., 1972) , 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 50 values of approximately 21 m (5.1 g/ml) and 73 m (17.8 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, 2006) . 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 1 (mnv-1) in the presence of ribavirin dropped about 10-fold after virus infection, but remained similar after 48 h of virus infection (chang and george, 2007) . 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 24 and 48 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, 2005) . 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 5 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, 2006; parker, 2005) . 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 3 (chang and george, 2007; leyssen et al., 2005) . 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 0.5 m and 0.1 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., 2000) . 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., 2000; day et al., 2005) . 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., 2001; contreras et al., 2002; day et al., 2005; lanford et al., 2001; severson et al., 2003) . 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 (1.89/1.66 versus 2.42/1.24). these results were not surprising since rna genomes mutate at average rates of 10 −3 -10 −5 per nucleotide copied during replication of rna viruses by the virus-encoded rna-dependent rna polymerase (rdrp) lacking proofreading functions (drake and holland, 1999) . 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 intracellular gtp levels via impdh inhibition. thus, we propose that one of the modes of action of ribavirin to elicit the antiviral effects against porcine nidoviruses occurs via gtp depletion, which may not work in concert with catastrophic mutations. although further studies based on in vivo assessment are needed to evaluate the efficacy and safety of ribavirin, the results presented here indicate that it is a good candidate of choice for antiviral approach against porcine nidoviral diseases. immune response and persistence of the porcine reproductive and respiratory syndrome virus in infected pigs and farm units epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview experimental infection of colostrum deprived piglets with porcine circovirus 2 (pcv2) and porcine reproductive and respiratory syndrome virus (prrsv) potentiates pcv2 replication functional properties of the 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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 (2010-0002318). key: cord-297531-et1sli23 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: 2017-10-20 journal: antiviral res doi: 10.1016/j.antiviral.2017.10.013 sha: doc_id: 297531 cord_uid: et1sli23 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 m27f, targeting to glycoprotein d (gd) of hsv-2, which also has cross-reactivity against hsv-1 gd. it has a high level of neutralizing activity against both hsv-1 and hsv-2, and binds to a highly conserved region (residues 292–297) within the pro-fusion domain of gd. it can effectively block hsv cell-to-cell spread in vitro. the preor post-attachment neutralization assay and syncytium formation inhibition assay revealed that m27f neutralizes hsv at the post-binding stage. moreover, therapeutic administration of m27f completely prevented infection-related mortality of mice challenged with a lethal dose of hsv-2. our newly identified epitope for the neutralizing antibody would facilitate studies of gd-based hsv entry or vaccine design, and m27f 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, 2013) , causing mucocutaneous lesions, keratitis, and encephalitis (dropulic and cohen, 2012) . there are two serotypes of hsv, hsv-1 and hsv-2, also known as human herpesvirus 1 and 2 (hhv-1 and hhv-2). the most common clinical sign of hsv-1 is herpes labialis, as well as ocular diseases, including conjunctivitis and keratitis (everett, 2014) . hsv-2 is the main cause of recurrent genital herpes, which is one of the most predominant sexually transmitted diseases, and hsv-2 infection of newborns during delivery is usually accompanied by a high mortality rate (cleary et al., 2005; domeika et al., 2010; overall, 1994) . moreover, epidemiological and biological studies have revealed that hsv-2 dramatically increases the risk of hiv infection (barnabas and celum, 2012; freeman et al., 2006; sartori et al., 2011) and may also act in conjunction with human papillomavirus (hpv) infection to increase the risk of invasive cervical carcinoma (smith et al., 2002; zhao et al., 2012) . according to world health organization (who) statistics, in 2012, there were 417 million people aged 15-49 years living with hsv-2 infection worldwide (looker et al., 2015) , and the newly infected population is estimated to be 23 million per year (looker et al., 2008) . 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-1. 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 1-260) carries the receptor binding sites, and the c terminus functions as the pro-fusion domain (residues 260-310), 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., 2004; fusco et al., 2005) . after binding to a cell surface receptor, either hvem or nectin-1, 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., 2007; eisenberg et al., 2012; fotouhi et al., 2008; nicola et al., 1998; whitbeck et al., 1999) . therefore, gd has been the predominant candidate for hsv vaccine or novel therapeutic strategies (awasthi and friedman, 2014; shin and iwasaki, 2013) . 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 1 to 23, 264 to 279, and 284 to 301 of the extracellular domain are major continuous antigenic determinants on gd (isola et al., 1989) . mabs recognizing epitopes located in these continuous determinant regions have no or low virus-neutralizing ability, except for the mab 1d3, which can neutralize viral infectivity by blocking gdreceptor interaction (cairns et al., 2014) . our current investigation found a mab, m27f that recognizes a new continuous epitope (residues 292 to 297) 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-1 and hsv-2, completely abrogated viral cell-to-cell spread, and inhibited syncytium formation in vitro. in addition, it also exhibited highly therapeutic effects in a hsv-2 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 10% fetal bovine serum (fbs) (thermo fisher scientific). hsv-1 f strain and hsv-2 333 strain were kindly provided by microorganisms and viruses culture collection center, wuhan institute of virology, cas (serial number: ivcas6.0182, ivcas6.0183). viruses were propagated in vero cells and cell lysate stocks were prepared as previously described (morrison and knipe, 1996) . virus titers were determined in vero cells (navarro et al., 1992) . 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. (2005) . (b) recognition region mapping of mabs m27f and 21c11. the truncated gd proteins expressed by pmal-c2x vector were presented as antigens. m27f and 21c11 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: syxk2014-0034). all animal experiments were approved by the institutional animal ethical committee of wiv, cas (approval: wiva23201403). 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-2 gd protein were prepared as described previously (evan et al., 1985) . in brief, the nucleotide sequence encoding the ectodomain (residues 1 to 318) plus the natural signal sequence of hsv-2 gd protein, gd1-318t, was cloned into the pet-32a vector (novagen) and expressed in escherichia coli bl21. the protein was purified under denaturing conditions in the presence of 8m urea by using a ninitrilotriacetic acid (nta) column and then dialyzed (pirestani et al., 2014) . the quantity of purified protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). balb/c mice (n = 2) were immunized with 60 μg of purified gd1-318t in complete freund's adjuvant (sigma). after 2, 4, and 6 weeks, the immunizations was repeated with gd1-318t conjugate in incomplete freund's adjuvant (sigma). fusion with sp2/0 myeloma cells was carried out by using standard methodology (de stgroth and scheidegger, 1980) . hybridomas culture supernatant was screened by enzyme-linked immunosorbent assay (elisa) and western blot analysis. totally 25 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-2 gd protein was dissected into seven truncated fragments, gd1-55t, gd35-95t, gd73-151t, gd125-194t, gd170-233t, gd213-278t and gd254-318t, with any two adjacent fragments sharing an overlap of 20-26 amino acids (fig. 1a) . amino acids 213 to 318 of the gd protein were further truncated into eight fragments, gd213-308t, gd213-298t, gd213-297t, gd213-296t, gd278-318t, gd288-318t, gd292-318t and gd293-318t (fig. 3a ). the nucleotides encoding the above fragments were pcramplified using the primer pairs listed in table s1 , cloned into pmal-c2x (neb) and then expressed in e. coli dh10β. for characterization of recognizing speciality, vero cells infected with hsv-1 or hsv-2 at an multiplicity of infection (moi) of 5 pfu per cell were harvested at 48 h post infection (p.i.) and rinsed with pbs. the protein samples were separated by 10% 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-2 gd protein: m27f and 21c11. 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-1 or hsv-2 at an moi of 5 pfu per cell. at 48 h p.i., the cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.05% triton x-100, and washed three times with phosphate-buffered saline (pbs). to characterize the recognizing speciality of m27f and 21c11, we immunostained cells with them respectively for 2 h at 37°c. pre-immune mouse serum and mouse immunoglobulin g (beyotime) were used as controls. all the antibodies were diluted 1:100 in blocking solution (5% 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., 2013) . in brief, vero cell were seeded into 12 well plates (costar) and incubated overnight to achieve confluent monolayers. m27f and 21c11 were serially diluted in dmem supplemented with 2% fbs and then combined with hsv-1 or hsv-2 (100 pfu/well) respectively (final concentration of m27f or 21c11 for hsv-1 ranged from 240 μg/ml to 0, and final concentration of m27f or 21c11 for hsv-2 ranged from 120 μg/ml to 0). pre-immune mouse serum was used as negative control. the virus-antibody mixtures were incubated for 1 h at 37°c with gentle rocking. after 1 h incubation, the mixtures were inoculated over vero cell monolayers in plates at 37°c for 2 h. after 2 h adsorption the cells were covered with overlay media with 1% methylcellulose to form plaques. the plaques developed after 72 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., 2011) . briefly, virusantibody mixture was incubated for 1 h at 4°c before inoculating over the vero cells (pre-attachment), while prechilled (4°c for 15 min) vero cells were infected with hsv-1 or hsv-2 (100 pfu/well) at 4°c for 1 h to allow virus adsorption before serial dilutions of antibodies were added (post-attachment). inoculated vero cells were then incubated for 2 h at 37°c. neutralization efficacy was determined after 72 h as described above for the standard neutralization assay. cell-to-cell spread assay was based on a previously described protocol (krawczyk et al., 2011) . bhk cells were grown on glass coverslips and incubated overnight to achieve confluent monolayers. cells were infected with hsv-1 or hsv-2 at an moi of 0.01 pfu per cell. after 2 h of adsorption at 37°c, the virus inoculum was removed and cells were washed twice with pbs and then incubated in dmem containing 2% fbs in the presence of antibodies, m27f (500 μg/ml) and 21c11 (500 μg/ml). mouse igg (500 μg/ml), or medium alone was used as control. to visualize the viral spread by immunofluorescence, after 48 h p.i., cells were fixed with 4% paraformaldehyde, permeabilized with 0.05% triton x-100, 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 ∼3000 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., 1992) . in brief, monolayers of vero cells grown in 24-well plates were infected with hsv-1 or hsv-2 at an moi of 5 pfu per cell. after 2 h of adsorption at 37°c, the virus inoculum was removed and cells were washed with pbs twice then incubated in dmem containing 2% fbs in the presence of antibodies, m27f (500 μg/ml) and 21c11 (500 μg/ml), mouse igg (500 μg/ml), or medium alone as a control. after 48 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 3 or more nuclei were analyzed. three independent experiments were calculated. mouse intravaginal hsv-2 challenge was established as previously described (cena-diez et al., 2016) . six-weeks-old female balb/c mice were rendered susceptible to infection by subcutaneous injection with 2.5 mg of medoxyprogesterone acetate (sigma) 5 days prior to challenge (parr et al., 1994) . mice were randomly assigned to five groups of 7 or 8 each. mice were anesthetized with pentobarbital sodium and intravaginally challenged with 1 × 10 6 pfu/20 μl of hsv-2. the infected mice were passively immunized by intravenous injection of 1, 3, 7 and 15 mg/kg of m27f at 24, 48, and 72 h after intravaginal hsv-2 challenge. pbs group was set as control. mice in each group were examined daily for symptoms of genital disease, body weight over 16 days, as well as mortality for 30 days. disease score was graded from 0 to 4: no apparent infection scored 0; genital erythema scored 1; moderate genital infection scored 2; purulent genital ulceration, hair loss and generally poor condition scored 3; severe ulceration of genital and surrounding tissue, and hind limb paralysis scored 4. mice that developing 20% 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 v6. statistical analysis was performed using graphpad prism 5. 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 < 0.05), ** (p < 0.01), or *** (p < 0.001). (original magnification, ×100) (c) and (d) virus neutralization assay. hsv-1 or hsv-2 (100 pfu/well) was incubated with indicated serially diluted antibodies m27f, 21c11 or preimmune serum. at 72 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-2 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 25 strains of positive hybridoma cells were obtained (table s2 ). epitope mapping of these mabs was determined using the expressed truncated gd proteins, including gd1-55t, gd35-95t, gd73-151t, gd125-194t, gd170-233t, gd213-278t and gd254-318t, with an overlap of 20-26 amino acids between each pair of adjacent fragments (fig. 1a) , by western blot analysis (data not shown). among these 25 mabs, m27f and another 14 mabs recognized truncated gd254-318t, which is located in the c-terminus (residues 260-310) of the gd ectodomain (fig. 1b) . in contrast, 21c11 (fig. 1b ) and the remaining 9 mabs recognized both nterminal fragments, gd1-55t and gd35-95t, suggesting that they recognized the overlap region (residues 35-55) of the two fragments. therefore, two kinds of mab, as represented by m27f and 21c11, recognizing different epitopes of hsv-2 gd, were chosen for further characterization. to characterize the specific recognition ability of m27f and 21c11, we first tested their ability to recognize denatured and native forms of hsv-1 gd and hsv-2 gd by western blot assay and ifa. western blot analysis revealed that m27f reacted with the expected gd band in the sample of both hsv-1 and hsv-2 infected vero cells, while 21c11 only recognized gd in the hsv-2 infected vero cells sample, but not the hsv-1 infected sample ( fig. 2a) . the ifa results demonstrated that m27f detected native forms of gd in the cells infected with either hsv-1 or hsv-2, while 21c11 only detected native gd in the cells infected with hsv-2 (fig. 2b) . these results suggested that m27f could recognize the denatured and native forms of gd of both hsv-1 and hsv-2, while 21c11 could only recognize the denatured and native forms of gd of hsv-2. to further confirm whether m27f and 21c11 could neutralize hsv infection, virus neutralization assays were performed. either hsv-1 or hsv-2 (100 pfu/well) were pre-incubated with serially diluted mabs, m27f and 21c11, before infection into vero monolayer cells. pre-immune mouse serum was used as negative control. neutralization assay demonstrated that m27f neutralized both hsv-1 and hsv-2 in a dosedependent manner, with about ∼60% hsv-1 infection and about ∼70% hsv-2 infection being inhibited at an antibody concentration of 120 μg/ml. in contrast, 21c11 demonstrated no neutralizing activity of hsv-1 and low neutralizing activity of hsv-2 infection groups (only about 20% virus infection being inhibited at a concentration of 120 μg/ ml) (fig. 2c and d) . these results suggested that m27f but not 21c11, is a neutralizing mab against hsv-1 and hsv-2 infection. as shown above, m27f had a high level of neutralization activity against both hsv-1 and hsv-2, and m27f recognized a linear epitope, gd254-318t, which is located in the c-terminus of the gd ectodomain. to identity accurately the epitope recognized by m27f, we constructed a series of further truncations of the c-terminal region (residues 213 to 318) of hsv-2 gd protein (fig. 3a) . by western blot analysis, gd213-297t but not gd213-296t that could be recognized by m27f, indicating that p297 is the last c-terminal amino acid critical for m27f binding (fig. 3b) . similarly, p292 is the first n-terminal amino acid critical for m27f binding. on this basis, 292 pnwhip 297 was considered to be the precise epitope for m27f binding. sequence alignment showed that these residues are highly conserved among hsv-1 and hsv-2 (fig. 3c) . these results explain why m27f recognized the gd protein of both hsv-1 and hsv-2. 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., 1991; hooks et al., 1976) . 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 m27f to inhibit cell-to-cell spread of hsv-1 and hsv-2 in vitro. confluent bhk cell monolayers were initially incubated with either hsv-1 or hsv-2 at an moi of 0.01 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 m27f or 21c11, and treatment with mouse igg or culture medium alone was performed as controls. after 48 h p.i., the transmission of virus was detected by immunostaining with mouse polyclonal antibody against gd. as shown in fig. 4a , m27f completely inhibited cell-to-cell spread of both hsv-1 and hsv-2, as evidenced by observing the limited fluorescence caused by virus infection. in contrast, neither 21c11 nor mouse igg could sufficiently inhibit transmission of hsv-1 or hsv-2, and extensive virus spread on bhk cell monolayer was observed by immunostaining. when the infected cells were quantified, the results clearly showed that m27f is able to inhibit cell-to-cell transmission in hsv-infected cells (fig. 4b) . to further characterize the mode of action of m27f, we analyzed whether this mab could inhibit receptor binding. we compared the neutralization efficacy of m27f when the antibody was added before (pre-attachment) or after (post-attachment) hsv virions interacted with vero cells. 21c11 was chosen as a negative control. by pre-or postattachment assays, when m27f was added before virus-host interaction (pre-attachment), about 60% hsv-1 infection was inhibited at a concentration of 120 μg/ml (fig. 5a) , about 65% hsv-1 infection was inhibited at a concentration of 120 μg/ml when m27f was added after hsv-1 virions interacted with vero cells (post-attachment) (fig. 5b) . almost equal neutralization efficacy of m27f was observed, irrespective fig. 4 . m27f blocks cell-to-cell spread of hsv-1 and hsv-2. (a) monolayers of bhk cells were infected hsv-1 or hsv-2 at an moi of 0.01 pfu per cell. cell monolayers were treated with an excess of antibodies, m27f and 21c11 (500 μg/ml each). mouse igg (500 μg/ml) or culture medium alone was performed as controls. at 48 h p.i., cells were fixed and observed by fluorescence microscopy. bar represents 100 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 < 0.05, ** p < 0.01, *** p < 0.001. of whether the antibody was added before or after hsv-1 virions interacted with vero cells. in contrast, the negative control, 21c11, had no neutralizing ability either before or after virions were incubated with cells. the hsv-2 infection group showed a similar result. about 80% hsv-2 infection was inhibited in the pre-attachment treatment (fig. 5c ) and about 90% hsv-2 infection was inhibited in the postattachment treatment with an antibody concentration of 120 μg/ml of m27f (fig. 5d ). in contrast, 21c11 had low (20% hsv-2 infection being inhibited) or no neutralizing ability in the pre-attachment or post-attachment treatments, respectively ( fig. 5c and d) . the existing data indicated that m27f does not interfere with the virus binding process, m27f 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 m27f might neutralize hsv entry at the post-binding stage, we asked whether m27f could inhibit the virus-induced membrane fusion process. monolayers of vero cells were infected with hsv-1 or hsv-2 at an moi of 5 pfu per cell. after 2 h of adsorption at 37°c, the virus inoculum was removed and cells were washed twice with pbs, the cells were then incubated with an excess of m27f, 21c11, mouse igg, or medium alone as a control. after 48 h, syncytium formation was observed by staining with he staining. as shown in fig. 6a , in both hsv-1 and hsv-2 infection groups, obvious syncytia with multinuclear cells were observed when infected cells were treated with preimmune serum, mouse igg or 21c11. however, when cells were incubated with m27f, syncytium formation was almost completely inhibited. the syncytium formation inhibition of vero cells with different treatment was quantified in fig. 6b . this result suggested that m27f indeed inhibited the hsv-induced membrane fusion. to investigate the protective efficacy of m27f in vivo, we exploited a fig. 5 . m27f neutralizes hsv-1 and hsv-2 at the post-binding stage. comparison of neutralization efficacy of m27f 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. 21c11 was used as a control. neutralization efficacies were determined after 72 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 3 independent experiments performed are shown. r. du et al. antiviral research 147 (2017) 131-141 lethal female balb/c mice model for hsv infection. to assess the therapeutic efficiency of m27f, mice were randomly assigned to five groups of 7 or 8 each. in pbs-treated group, intravaginal infection of hsv-2 (1 × 10 6 pfu/20 μl) of six-weeks-old female balb/c mice resulted in rapid progressive disease (fig. 7a ) and steady decrease of body weight from day 4 post infection (fig. 7b) , with a median survival time of 10 days (fig. 7c) . the experimental groups of balb/c mice were treated intravenously with 1, 3, 7, or 15 mg/kg of antibody at 24, 48 and 72 h after intravaginal hsv-2 challenge. as shown in fig. 7 , mice receiving 1 mg/kg of m27f were slightly protected against lethal infection by hsv-2, the body weight of this group decreased as fast as the pbs-treated group, with a median survival time of 2 days longer than those of control mice treated with pbs. however, mice treated with 3 mg/kg or higher concentrations of m27f were completely protected from lethal hsv-2 infection (fig. 7c) . no symptom of hsv-2 infection (fig. 7a ) or loss of body weight was observed among these groups (fig. 7b) . 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, mc23 (which recognizes residues 213 and 216), dl11 (which recognizes residues 132 and 140), mc5 (which recognizes residues 75-79) and mc2 (which recognizes residue 246) have been reported previously to be neutralizing mabs that recognize discontinuous conformational epitopes. lazear et al., 2012; muggeridge et al., 1990; nicola et al., fig. 6 . m27f inhibits syncytium formation of hsv-1 and hsv-2. (a) vero cells were infected with hsv-1 or hsv-2 at an moi of 5 pfu per cell and then incubated in dmem containing 2% fbs in the presence of antibodies, m27f (500 μg/ml) and 21c11 (500 μg/ml). mouse igg (500 μg/ml), or medium alone were used as control. after 48 h, cells were fixed and then stained with he staining. the inhibition of syncytium formation was observed by light microscopy. bar represents 100 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 3 or more nuclei were analyzed. data were representative of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. 1998; whitbeck et al., 1999) . 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 1d3 (which recognizes residues 10 to 20), which can neutralize viral infectivity by blocking gd-receptor interaction (cairns et al., 2014) . in this study, we found a novel mab, m27f, to be another virusneutralizing mab whose epitope is located within a continuous antigenic determinant. epitope mapping assays indicated that m27f recognized conserved residues 292 to 297 which located in the c-terminal pro-fusion domain (residues 260-310) of gd ( figs. 1 and 3) . sequences alignment analysis showed that this epitope is highly conserved among a broad range of type-1 and type-2 hsv strains (data not shown). this may also explain why the recognition specificity of m27f was type-common. it is likely that m27f can be efficacious among divergent isolates yet the breadth of m27f neutralizing activity against a panel of hsv-1 and -2 isolates in vitro need to be performed. in addition, the resistance evaluation of m27f and alanine scanning of 292-297 should also be carried out in the future. the recognition specificity of m27f was type-common, and m27f had a high level of hsv virion neutralizing activity (fig. 2) . the epitopes of three reported type-common mabs, bd78 (which recognizes residues 262 to 272), dl6 (which recognizes residues 272 to 279), and bd66 (which recognizes residues 280 to316), were continuous and also located in the c-terminal pro-fusion domain of gd. however, they had no neutralization activity against both hsv-1 strain kos and hsv-2 strain 333 (isola et al., 1989; lazear et al., 2012) . previously, hooks and his colleagues found hsv-1, human and murine cmv and vaccinia could spread in the presence of neutralizing antibodies generated in rabbits (hooks et al., 1976) . co et al. humanized two murine monoclonal antibodies against hsv gb and gd, which had been shown to neutralizing hsv-1 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., 1991) . our research showed that m27f is able to inhibit cell-to-cell spread of hsv-1 and hsv-2 in vitro (fig. 4) , which is an efficient way for hsv to bypass the host immune system through the direct transmission adjacent cells. to our knowledge, m27f is the first epitope-mapped neutralizing mab against hsv gd reported to be capable of inhibiting cell-to-cell spread of hsv-1 and hsv-2. 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 (s2) and globular (s1) 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 gp120 to the cd4 receptor (wibmer et al., 2015; zeng et al., 2006) . the above-mentioned neutralizing mabs mc23 and dl11 neutralize virus by blocking binding of gd to both receptors, hvem and nectin-1. 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., 2003) . mab 44d11 against the f 2 subunit of the helicoverpa armigera nucleopolyhedrovirus f protein neutralizes virus entry by inhibiting membrane fusion (zou et al., 2016) . our data from pre-attachment and post-attachment neutralization assays indicated that m27f does not interfere with the virus binding process; instead, it might neutralize hsv entry at the post-binding stage (fig. 5) . by observing hsv-induced membrane fusion following treatment with m27f, we determined that m27f could almost completely inhibit syncytium formation (fig. 6) , which further confirms that m27f could inhibit the membrane fusion event rather than the virus binding process. consistent with current findings, earlier studies (cocchi et al., 2004; fusco et al., 2005) established that the profusion domain (residues 260-310) is required for viral infectivity and fusion but not for receptor binding and the substitutions of some pro residues in pfd (amino acids 288, 289, 291, and 292) affected steps in entry and fusion. however, the detailed mechanism of how m27f inhibits the hsv fusion process needs further study. based on the high level of hsv virion neutralizing activity, we also investigated whether application of m27f could confer protection in vivo by exploiting a lethal hsv-2 intravaginally infected female balb/c mouse model to evaluate the potency of m27f as therapeutic alternative to counteract hsv infection. our data demonstrated that mab m27f conferred protection in a dose-dependent manner and that 3 mg/kg or more of m27f provided full protection (fig. 7) . consistently, intracutaneously injection human recombinant neutralizing mab hsv8 specific for gd (type-common and its epitope located in residues 234 to 275) can protect mice effectively when administered systemically zeitlin et al., 1996) . passive immunization of immunocompetent animals with gd specific mab hd1 (a type-common fig. 7 . m27f protects mice from lethal hsv-2 infection. (a) symptoms of genital disease in each group was scored daily as described in the text for 16 days after intravaginally hsv-2 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 = 8 animals per group for pbs; n = 7 for all other groups) (c) survival of mice in each group monitored for indicated days. (n = 8 animals per group for pbs; n = 7 for all other groups). data were analyzed by log-rank (mantel-cox) test and gehan-breslow-wilcoxon test. * p < 0.05, *** p < 0.001. neutralizing mab, and its discontinuous epitope located in residues 1 to 234) postexposure at appropriate times demonstrated protection against 3 hsv-1 strains (htz, mp, and a mutant strain mp) and 1 hsv-2 strain g -induced neurological disease (dix et al., 1981; pereira et al., 1980) . furthermore, intravaginally administered murine anti-gb mab post hsv-1 infection conferred full protection in an immunodeficient non-obese diabetic (nod)/severe combined immunodeficiency (scid) mouse model (krawczyk et al., 2011) . 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 m27f targeting a new continuous epitope (residues 292 to 297) within the pro-fusion domain has a high level of 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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/10.1016/j.antiviral.2017.10.013. key: cord-266585-jfjrk9gy 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 (nsp13) is lethal to the coronavirus infectious bronchitis virus in cultured cells date: 2007-02-05 journal: virology doi: 10.1016/j.virol.2006.08.020 sha: doc_id: 266585 cord_uid: jfjrk9gy 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 15526, causing arg-to-pro mutation at amino acid position 132 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 g15526c mutation demonstrated that this mutation may block the transcription of subgenomic rnas. substitution mutation of the arg132 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 arg132 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 4330 and 9230, respectively, causing glu–gln and gly–glu mutations in or near the catalytic centers of the papain-like (nsp3) and 3c-like (nsp5) 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 2003, a novel coronavirus (sars-cov) was identified as the causative agent of severe acute respiratory syndrome (sars) (marra et al., 2003; rota et al., 2003) . 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., 2000; casais et al., 2001 casais et al., , 2003 casais et al., , 2005 coley et al., 2005; hodgson et al., 2006; koetzner et al., 1992; masters and rottier, 2005; sanchez et al., 1999; youn et al., 2005a youn et al., , 2005b yount et al., 2000 yount et al., , 2002 yount et al., , 2003 yount et al., , 2005 . avian infectious bronchitis virus (ibv), a group 3 coronavirus, causes an acute and contagious disease in chickens with a significant impact on the poultry industry worldwide. ibv contains a 27.6 kb single-stranded, positive-sense rna genome. in the virus-infected cells, six mrna species, including the genome-length mrna 1 and five subgenomic mrnas (mrna 2-6), are produced by a discontinuous rna transcription mechanism. each mrna species possesses a 64 nucleotides leader sequence derived from the 5′ end of the virology 358 (2007) 136 -147 www.elsevier.com/locate/yviro genome (boursnell et al., 1987) . subgenomic mrnas 2, 3, 4, and 6 encode the four structural proteins, i.e., spike glycoprotein (s), envelope protein (e), membrane protein (m), and nucleocapsid protein (n). the 5′ two-third region of mrna 1 comprises two large orfs, 1a and 1b, and encodes two polyproteins. the two polyproteins are proteolytically cleaved by two virus-encoded proteinases, the papain-like and 3c-like proteinases, into 15 functional proteins (nsp2-nsp16) liu, 1998a, 1998b; lim et al., 2000; liu et al., 1995 liu et al., , 1997 liu et al., , 1998 ng and liu, 1998 xu et al., 2001) . compared to other coronaviruses, nsp1 is absent in ibv but nsp2 is considerably larger liu, 1998a, 1998b; liu et al., 1995) . 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 (g15526c) point mutation at nucleotide position 15,526 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 arg132-pro mutation in a domain with unknown function within the helicase protein (nsp13), it implies that this region might play certain roles in the functionality of the helicase protein. on the contrary, a g-c (g4330c) and a g-a (g9230) point mutations at nucleotide positions 4330 and 9230, respectively, causing glu-gln and gly-glu mutations in or near the catalytic centers of the papain-like (nsp3) and 3c-like (nsp5) 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 g15526c mutation demonstrated that this mutation blocks the transcription of subgenomic rnas. substitution mutation of the arg132 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 arg132 residue to a leu, which is conserved in most of other coronaviruses at the same position, reduced the viral recovery rate of the in vitrofig. 1 . 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 3a, 3b, 5a, and 5b, and the 5′-and 3′-utr are shown. also shown are the regions of the five rt-pcr fragments, the t7 promoter at the 5′-end of fragment a, and the 30 as at the 3′-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 1-5751 (lane 2), 5752-8693 (lane 3), 8694-15,520 (lane 4), 15,521-20,900 (lane 5) , and 20,901-27,608 (lane 6), respectively, were obtained by digestion of corresponding plasmid dna with either bsmbi or bsai, purified from agarose gel, and analyzed on a 0.8% agarose gel (lanes 2-6). equal amounts of the purified fragments were ligated using t4 dna ligase (lane 7) and analyzed on a 0.4% 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 0.8% agarose gel (lane 10). lanes 1, 7, and 9 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 (p65) (shen et al., 2003 (shen et al., , 2004 fang et al., 2005) . to facilitate the assembly of the full-length cdna in vitro, restriction sites for either bsmbi or bsai were introduced into both the 5′ and 3′ ends of the fragments (fig. 1a) . in fragment a, a 19-nucleotide sequence corresponding to the t7 rna promoter (table 1) was inserted into the 5′ end of the ibv genome to facilitate in vitro transcription using the t7 polymerase (fig. 1a) . the primers used to amplify these fragments are listed in table 1 . 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 pkt0 which contains a bsmbi site 400 bp upstream of the t7 promoter sequence (fig. 1a) . 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 2 . the five fragments were then prepared by digestion of the corresponding constructs with either bsmbi or bsai and purified (fig. 1b, lanes 2-6) . the full-length clone was made by ligation of the purified fragments in vitro (fig. 1b, lane 8) and used as the template for in vitro transcription. the fulllength in vitro-synthesized transcripts were generated using the mmessage mmachine t7 kit (ambion, austin, tex) (fig. 1b , lane 10). 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., 2001; youn et al., 2005a youn et al., , 2005b yount et al., 2000 yount et al., , 2002 , the n transcripts were generated from a linearized pkt0-ibvn construct containing ibv n gene and the 3′-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 (p65, accession no. dq001339) shows nucleotide changes at 16 positions (table 2 ). among them, 11 a unique mutations found only in the cloned fragments. b same as the published sequences but different from p65. caused unique amino acid changes (table 2) . 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, g4330c, g9230a, and g15526c, 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., g4330c containing g-c mutation at nucleotide position 4330 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 2 days post-electroporation, a typical cpe of the vero cell-adapted ibv, the formation of giant syncytial cells (fang et al., 2005) , was observed in cells transfected with transcripts generated from cdna clones ribv, g4330c, and g9230a. cpe was extended to almost the whole monolayers at 3 days post-electroporation ( fig. 2a) . no cpe was observed in cells transfected with transcripts generated from clone g15526c (fig. 2a) . rt-pcr analysis of the subgenomic mrna 5 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. 2b ). 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 8 amino acid mutations (table 2 ). to test if these mutations may affect the growth properties and genetic stability of the rescued virus, ribv was propagated on vero cells for 5 passages, and the plaque sizes and growth kinetics were determined and compared with wild type ibv p65. in cells infected with ribv, the average plague size is 0.56 ± 0.028 mm, which is slightly smaller than the average plaque size of 0.68 ± 0.034 mm in cells infected with wild type ibv (fig. 3a) . analysis of the growth curves demonstrated that ribv exhibited very similar growth properties as the wild type virus (fig. 3a) . 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. 3b ), and s, n, and m proteins ( fig. 3c ) in cells infected with wild type and ribv, respectively, at 24 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. 3c ). 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. 3c ). 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 g15526c 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 g15526c mutant transcripts at 24 and 48 h post-electroporation, respectively. reverse transcription was performed by using equal amount of rna and the sense-primer ibv14931-f (5′-14,931 gct-tatccactagtacatc 14,949 -3′), and pcr was carried out by using the sense-primer ibv14931-f and the antisense-primer ibv15600-r (5′-15,600 cttctcgcacttctgcacta-gca 15,578 -3′). if replication of viral rna occurred, a 670 bp pcr fragment would be expected. as shown in fig. 4a , rt-pcr fragments amplified from negative strand rna templates were obtained from cells transfected with both wild type (lanes 2 and 4) and the mutant transcripts (lanes 3 and 5). 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. 4a, lane 6) , 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 g15526c mutant transcripts was carried out by real-time pcr. at 24 and 48 h post-electroporation, transcription of the negative rna in cells electroporated with wild type transcripts was 24.76-and 945.54-fold, respectively, higher than that in cells electroporated with the g15526c 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. 3 . analysis of the growth properties of wild type (p65) and ribv. (a) plague sizes and one-step growth curves of wild type and ribv. monolayers of vero cells on a 6-well plate were infected with 100 μl of 1000-fold diluted virus stock and cultured in the presence of 0.5% carboxymethy cellulose at 37°c for 3 days. the cells were fixed and stained with 0.1% toluidine. to determine the one-step growth curves of wild type and ribv, vero cells were infected with the viruses and harvested at 0, 4, 8, 12, 16, 24, and 36 h post-inoculation, respectively. viral stocks were prepared by freezing/thawing of the cells three times, and tcid50 of each viral stock was determined by infecting five wells of vero cells on 96-well plates in triplicate with 10-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 1% agarose gel and transferred to a hybond n+ membrane. viral rnas were probed with a dig-labeled dna probe corresponding to the 3-end 680 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 1) and ribv (lane 2) were harvested, lysates prepared and separated on sds-10% 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 2 days postelectroporation was used in the rt reaction with the sense-primer ibv-leader (5′-26 ctattacactagccttgcgct 46 -3′) for the detection of negative-stranded sgrna, and the antisenseprimer ibv24803-r (5′-24,803 ctctggatccaataacc-tac 24,784 -3′) for the detection of positive-stranded sgrna. the two primers were then used for pcr. if transcription of subgenomic mrnas did occur, a 415 bp pcr product corresponding to the 5′-terminal region of the subgenomic mrna 4 and a 1010 bp fragment corresponding to the 5′terminal region of the subgenomic mrna 3 would be expected. as shown in fig. 4b , a dominant 415 bp band and a weak 1010 bp band were observed in cells electroporated with wild type full-length transcripts at 2 days post-electroporation (lanes 2 and 5). sequencing of the pcr fragments confirmed that they represent the correct sequences of the corresponding regions of the subgenomic mrnas 3 and 4, respectively. however, the same pcr products were not detected in cells electroporated with the mutant transcripts (fig. 4b, lanes 3 and 6) . as a negative control, the amplified fragments were not detected in cells without electroporation (fig. 4b, lane 4) . the failure to detect both negative-and positive-stranded sgrnas in cells transfected with the mutant transcripts show that the g15526c mutation leads to the disruption of subgenomic rna transcription. to further demonstrate that the failure to rescue infectious virus from the g15526c mutant transcripts is due to a defect in subgenomic rna transcription, the full-length clones with and without the g15526c mutation were used to generate recombinant ibv expressing the enhanced green fluorescent protein (egfp) by replacing the 5a 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 2 days post-electroporation, single cpe with the expression of egfp was observed in cells transfected with the full-length transcripts without the g15526c mutation vero cells electroporated with in vitro-synthesized transcripts derived from the in vitro assembled full-length clones containing egfp either with or without g15526c mutation. phase-contrast and fluorescent images were taken 2, 3, and 5 days post-electroporation, respectively. (fig. 4c) . gradually increased cpe and fluorescent cells were observed from 3 to 5 days post-electroporation (fig. 4c) . at 5 days post-electroporation, cpe and fluorescent cells were extended to almost the whole monolayer (fig. 4c) . 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 g15526c mutation, neither cpe nor cells expressing egfp were observed (fig. 4c) , demonstrating that g15526c 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 g15526c mutation blocks subgenomic rna transcription. mutational analysis of the r132 residue of the helicase protein g15526c mutation resulted in the substitution mutation of the r132 with a pro (r132p) of the helicase protein. sequence comparison of the ibv helicase protein with other known coronaviruses showed that r132 residue is located adjacent to a conserved motif (fig. 5a) . in all sequenced coronaviruses, only ibv has a charged amino acid (r132) at this position (fig. 5a) . to assess if a positive charge amino acid at this position is essential for the function of the protein, mutation of r132 to a lys (r132k) 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 r132 to a leu (r132l) was also included. in vitro full-length transcripts containing the r132k and r132l mutations were electroporated into vero cells. as shown in fig. 5b , transcripts generated from wild type (r132) and r132k mutant constructs showed very similar infectivity after introduction into vero cells. typical cpe was observed in large areas of the monolayers at 3 days post-electroporation (fig. 5b) , and recombinant viruses were recovered. however, r132l 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 3 days post-electroporation (fig. 5b) . the growth properties of the r132k and r132l mutant viruses on vero cells were tested by analysis of plaque sizes and growth curves of passage 5 mutant viruses. compared to cells infected with wild type recombinant virus (average plaques size is 0.56 ± 0.028), plaques with similar size were observed in cells infected with the r132k mutant virus (fig. 6a) . the average plaque size in cells infected with r132k mutant virus is 0.59 ± 0.029 mm. in cells infected with the r132l mutant virus, much smaller-sized plaques were observed (fig. 6a) . the average plaque size in cells infected with this mutant virus is 0.24 ± 0.017 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. 6a) . the r132k and r132l 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 24 h post-infection (fig. 6b) . similarly, western blot analysis of cells infected with ribv, r132k, and r132l mutant viruses showed that similar amounts of s, n, and m proteins were detected at 24 h post-infection (fig. 6c, lanes 1-3) . when probing with anti-n protein antibodies, other species migrating faster than the full-length products on sds-page were also observed (fig. 6c) . 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. 6c) . the genetic stability of r132k and r132l mutant viruses was tested by propagation of the viruses on vero cells for 5 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. (2000) , are a rapid and reliable approach to construct infectious clones from large rna viruses. it has been successfully used to construct infectious fig. 6 . analysis of the growth properties of ribv, r132k, and r132l mutant viruses. (a) plague sizes and one-step growth curves of ribv, r132k, and r132 mutant viruses. monolayers of vero cells on a 6-well plate were infected with 100 μl of 100-fold diluted virus stock and cultured in the presence of 0.5% carboxymethy cellulose at 37°c for 3 days. the cells were fixed and stained with 0.1% toluidine. to determine the one-step growth curves of ribv, r132k, and r132l mutant viruses, vero cells were infected with the viruses and harvested at 0, 4, 8, 12, 16, 24 , and 36 h postinoculation, respectively. viral stocks were prepared by freezing/thawing of the cells three times, and tcid50 of each viral stock was determined by infecting five wells of vero cells on 96-well plates in triplicate with 10-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, r132k, and r132l mutant viruses. ten micrograms of total rna extracted from vero cells infected with ribv, r132k, and r132l mutant viruses, respectively, was separated on 1% agarose gel and transferred to a hybond n+ membrane. viral rnas were probed with a dig-labeled dna probe corresponding to the 3-end 680 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 r132 mutant viruses. vero cells infected with wild type recombinant ibv (lane 1), r132k (lane 2), and r132l (lane 3) were harvested, lysates prepared, and separated on sds-10% 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., 2005a (youn et al., , 2005b yount et al., 2000 yount et al., , 2003 . 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 15,526 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 arg132-pro substitution in a domain within the helicase protein (nsp13) 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 nsp13 helicase protein. these include rna and dna duplex-unwinding activities, ntpase and dntpase activities, and an rna 5′-triphosphatase activity that might be involved in the formation of the 5′-cap structure of viral rnas (ivanov et al., 2004) . 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 1 to 77, and a c-terminal helicase domain covering the c-terminal part of the protein from amino acids 279 to the cterminal end (ivanov et al., 2004) . a ser-pro substitution located immediately downstream of the putative zinc binding domain of nsp10, the equivalent rna helicase protein in equine arteritis virus (eav), caused defect in subgenomic mrna transcription (van dinten et al., 1997 (van dinten et al., , 2000 . more detailed analysis of the zinc binding domain of nsp10 from eav and nsp13 from human coronavirus 229e by mutagenesis studies showed that this domain could modulate the enzymatic activities of the helicase domain (seybert et al., 2005) . 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 g15526c (r132p) 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., 1997 (van dinten et al., , 2000 . mutation of the arg132 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 arg132 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 r132 residue is located 57 amino acids downstream of the last his (h75) residue in the putative zinc binding domain (fig. 5a) and 147 amino acids upstream of the helicase motif 1 (seybert et al., 2005) . so far, no functional domain has been found in this region of the helicase protein. why the r132p 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, r132 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. r132p mutant virus shares certain phenotypic similarity to the ser-pro mutant eav, such as the absence of subgenomic mrna transcription (van dinten et al., 1997 (van dinten et al., , 2000 , suggesting that the two mutations might disrupt a similar function of the helicase protein. however, as biochemical characterization of the effect of r132p mutation on the enzymatic activities of ibv nsp13 helicase protein is currently lacking, it would be difficult to draw a conclusion that the two mutants share mechanistically similar characteristics. second, r132 is located in a region with a high degree of amino acid conservation in all known coronaviruses. since the main defect of r132p 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 g433c (e-q) point mutation near the catalytic center of the plp domain in nsp3 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 g9230a (g-e) point mutation in the 3clp 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 37°c in minimal essential medium (mem) supplemented with 10% fetal bovine serum (fbs), penicillin (100 units/ml), and streptomycin (100 μg/ml). a vero cell-adapted ibv beaudette strain (65 passages on vero cells (p65)) (shen et al., 2003 (shen et al., , 2004 fang et al., 2005) 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 p65 at a multiplicity of approximately 1. 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-5753r, ibv-8694r, ibv-15532r, ibv-20930r, and ibv-27608r (table 1) . 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 pkt0 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 0.8% 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 t4 dna ligase at 4°c overnight. the two reaction mixtures were then mixed and further ligated at 4°c overnight. the final ligation products were extracted with phenol/chloroform/isoamyl alcohol (25:24:1), precipitated with ethanol, and detected by electrophoresis on 0.4% agarose gels. full-length transcripts were generated in vitro using the mmessage mmachine t7 kit (ambion, austin, tx) according to the manufacturer's instructions with certain modifications. briefly, 30 μl of transcription reaction with a 1:1 ratio of gtp to cap analog was sequentially incubated at 40.5°c for 25 min, 37.5°c for 50 min, 40.5°c 25 min and 37.5°c for 20 min. the n transcripts were generated by using a linearized pkto-ibvn containing ibv n gene and the 3′-utr region as templates. a 1:2 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 90% confluence, trypsinized, washed twice with cold pbs, and resuspended in pbs. rna transcripts were added to 400 μl of vero cell suspension in an electroporation cuvette and electroporated with one pulse at 450 v, 50 μf with a bio-rad gene pulser ii electroporator. the transfected vero cells were cultured overnight in 1% fbs-containing mem in a 60 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 24 or 48 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 1, and total rna was extracted from the infected cells. ten micrograms of rna was added to a mixture of 1× mops, 37% formaldehyde, and formamide and incubated at 65°c for 20 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 50°c in hybridization oven overnight. membranes were washed 3 times for 15 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 1. total proteins extracted from the infected vero cells were lysed with 2× sds loading buffer in the presence of 200 mm dtt plus 10 mm of iodoacetamide and subjected to sds-page. proteins were transferred to pvdf membrane (stratagene) and blocked overnight at 4°c in blocking buffer (5% fat free milk powder in pbst buffer). the membrane was incubated with 1:2000 diluted primary antibodies in blocking buffer for 2 h at room temperature. after washing three times with pbst, the membrane was incubated with 1:2000 diluted anti-mouse or anti-rabbit igg antibodies conjugated with horseradish peroxidase (dako) in blocking buffer for 1 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 100 μl of 100-fold diluted virus stock. after 1 h of incubation at 37°c, cells were washed twice with pbs and cultured in 3 ml of mem containing 0.5% carboxymethy cellulose for 3 days. the cells were fixed and stained with 0.1% 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 50% tissue culture infection dose (tcid50) 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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 orf1a of the coronavirus ibv and determination of the cterminal cleavage site of an 87 kda protein characterization of the two overlapping papainlike proteinase domains encoded in gene 1 of the coronavirus infectious bronchitis virus and determination of the c-terminal cleavage site of an 87 kda protein identification of a novel cleavage activity of the first papain-like proteinase domain encoded by open reading frame 1a of the coronavirus avian infectious bronchitis virus and characterization of the cleavage products identification, expression and processing of an 87k polypeptide encoded by orf1a of the coronavirus infectious bronchitis virus proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10 kda polypeptide and determination of its cleavage sites proteolytic mapping of the coronavirus infectious bronchitis virus 1b polyprotein: evidence for the presence of four cleavage sites of the 3c-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 24 kda polypeptide processed from the coronavirus infectious bronchitis virus 1a polyprotein by the 3c-like proteinase and determination of its cleavage sites further characterization of the coronavirus infectious bronchitis virus 3c-like proteinase and determination of a new cleavage membrane association and dimerization of a cysteinerich, 16-kda polypeptide released from the c-terminal region of the coronavirus infectious bronchitis virus 1a 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 3b gene: functional characterization of the 3b 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 1b region of the avian coronavirus infectious bronchitis virus 1a/1b 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 5a 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 a59 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 03/1/22/17/220), agency for science, technology and research, singapore. key: cord-305496-t8ykkekl 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-2 and methods for detection of neutralizing antibody by focus forming assay date: 2020-08-21 journal: biorxiv doi: 10.1101/2020.08.20.259838 sha: doc_id: 305496 cord_uid: t8ykkekl the sars-cov-2 outbreak and subsequent covid-19 pandemic have highlighted the urgent need to determine what cells are susceptible to infection and for assays to detect and quantify sars-cov-2. furthermore, the ongoing efforts for vaccine development have necessitated the development of rapid, high-throughput methods of quantifying infectious sars-cov-2, as well as the ability to screen human polyclonal sera samples for neutralizing antibodies against sars-cov-2. to this end, our lab has adapted focus forming assays for sars-cov-2 using vero ccl-81 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-2 neutralizing antibody titer in human, non-human primate, and mouse polyclonal sera following sars-cov-2 exposure. additionally, we describe the viral growth kinetics of sars-cov-2 in a variety of different immortalized cell lines and demonstrate via human ace2 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-2 infection, with particular 72 uncertainty including if higher ace2 expression coincides with increased risk for heightened 73 infection. furthermore, it is unclear which cell types may be suitable for the successful evaluation 74 of antiviral and therapeutic drugs for in vitro screening before in vivo evaluation. further 75 characterization of permissible cell types is necessary to improve our understanding of sars-76 cov-2 pathogenesis and to develop therapeutic strategies for the treatment of it has also been noted that following infection with sars-cov-2, people typically 78 seroconvert 20 days following symptom onset, and there is increasing evidence suggesting that 79 development of a neutralizing antibody response is a correlate of protection in patients recovered 80 from . the covid-19 pandemic has highlighted the necessity for testing for 81 neutralizing antibodies. as sars-cov-2 infection can have a range of manifestations from 82 asymptomatic to fatal multiple organ failure [23] [24] [25] [26] [27] , antibody testing and serological surveys are 83 a critical tool for determining prior infection status and seroprevalence in a population . it is also 84 the goal of many candidate sars-cov-2 vaccines to induce neutralizing antibodies targeting the 85 viral spike protein, the major antigenic determinant of sars-cov-2 [28] . 86 for understanding the antibody response, assays that measure neutralizing antibody titer 87 are considered the gold standard. one such tool for evaluating neutralizing antibody response is a 88 plaque/focus neutralization reduction test (prnt/frnt), which evaluates the ability of polyclonal 89 sera samples to prevent or reduce infection of a cell monolayer in vitro. previously, for sars-90 cov-2, only prnt assays-which rely on the ability of virus to lyse infected cells and thus can 91 take 48-96 hours to develop-have been used in the assessment of the neutralizing antibody 92 response. it is not well known whether an frnt-which uses an immunostaining protocol to 93 detect virus and does not depend on cell lysis, and thus is often more rapid-is amenable for 94 detection of sars-cov-2 neutralizing antibody. 95 in this study, we describe the growth kinetics of sars-cov-2 in multiple cell types and 96 the methods our laboratory has used to optimize a sars-cov-2 focus forming assay (ffa) to 97 improve sensitivity and specific detection. by characterizing the growth kinetics of sars-cov-2 98 on a variety of immortalized and primary cell lines, we have demonstrated which of these cell lines 99 is susceptible to infection by sars-cov-2.we also demonstrate that the ffa can be adapted to 100 measure the neutralization capacity of polyclonal sera in an frnt. this high throughput frnt 101 assay can be applied to sera from both animal models of sars-cov-2 infection, as well as human 102 sars-cov-2 infected patients, and can serve as a useful assay for describing the kinetics of the 103 neutralizing antibody response to sars-cov-2. additionally, we have compared the expression 104 of ace2 and sars-cov-2 spike on these cell lines to determine how spike expression correlates 105 with susceptibility. the tools developed in this study have practical applications in both the basic 106 science and translational approaches that will be critical in the ongoing effort to slow the covid-107 19 pandemic. based on our previous work optimizing the ffa for wnv, [29] we proceeded to adapt the 113 ffa for the detection of infectious sars-cov-2. along with spot number, the spot size and 114 border definition provide valuable information on possible differences in viral strains. as we have 115 observed that the foci morphology, as well as spot number, can vary dramatically under different 116 growth conditions, we sought to test different growth conditions and cell lines to determine the 117 optimal conditions for sars-cov-2 viral titration. this goal was guided by previous studies that 118 have suggested that the use of vero cells from varying origins can impact viral titer [30, 31] . using 119 both vero ccl-81 (atcc® ccl-81™, referred to in this text as vero who) and vero e6 (vero 120 1008, atcc® crl-1586™) cell lines, we determined if differences in foci number or size 121 occurred to decide if one cell line was superior for titration by ffa (figure 1a-1c) . although 122 many laboratories utilize vero e6 cells for viral titer measurements of sars-cov-2 [32, 33] , in 123 our laboratory, vero e6 cells typically resulted in about two-fold lower foci formation relative to vero who cells (figure 1a-c) . figure 1a is a representative image of an ffa showing the 125 viral titration on both the vero e6 and whos for both ~50 ffu and ~200 ffu when identical 126 numbers of cells are seeded per well. we noted that at identical higher dilutions of sars-cov-2 127 virus stocks, vero who cells develop ~55 individual foci per well, whereas vero e6 cells develop 128 ~27 foci per well ( figure 1a) . the same pattern was observed at lower dilutions of virus, with 129 ~200 foci formation on vero who cells yielding only ~100 foci on vero e6 cells ( figure 1a ) 130 the quantification of this difference in the vero who and vero e6 cell type is shown in figure 131 1b. interestingly, when we compared this observation to the genome copy number by 132 we noted that vero e6 cells tended to produce significantly more virus across all 24, 48, and 72-133 hour timepoints (p = 0.0064) ( figure 1c) . it is possible that the discrepancy between the ffa and 134 qrt-pcr data could be due to vero who cells producing fewer genome copies yet more 135 infectious virus than e6 cells, while e6 cells sustain more viral replication but yield less infectious 136 virus. while we did not see any differences in foci morphology between the two vero cell lines 137 we used, the significant difference in the number of foci observed between vero e6 and vero 138 who (p < 0.0001) suggests that vero who cells record higher viral titers than the vero e6 cells 139 for sars-cov-2 ( figure 1b) . 140 141 in order for sars-cov-2 to form distinct foci, it is critical to plate the optimal cell density. 143 we examined the impact of cell density on foci formation for both vero who and vero e6 cells 144 by plating identical dilutions of sars-cov-2 virus stocks on 96-well plates seeded with differing 145 numbers of who or e6 cells (3 × 104, 1.5 × 104 or 3 × 104 cells/well) one day prior to infection 146 of the cell monolayer. at these concentrations, on the day of infection, 3×104, 1.5×104 and 3×104 147 cells/well resulted in monolayers that were 70, 80 and 90 percent confluent respectively for both 148 cell lines tested. figure 1d is a representative image of the focus forming assay showing the foci 149 formations arising from different cell concentrations plated for both the vero e6 and whos. the 150 quantification of the spot counts for vero who cells is shown in figure 1e . the quantification 151 of the spot counts for vero e6 cells is shown in figure 1f . for both the vero whos and e6 cells, 152 we observed a significant increase in the number of foci formed when either 1.5 × 104 (e6 p = 153 0.0480, who p = 0.0094) or 3 × 104 (e6 p = 0.0057, who p = 0.0024) were seeded per well 154 compared to 3 × 103. however, there was no significant increase in foci formation when increasing the cell density from 1.5 × 104 cells/well to 3 × 104 cells/well. thus, while the vero e6 cells 156 fostered lower foci numbers at each confluency as compared to the who cells, viral titers were 157 not significantly different at 1.5 × 104 cells/well or 3 × 104 cells/well irrespective of whether the 158 vero who (figure 1e ) or vero e6 ( figure 1f ) cell line was seeded for the assay. we have 159 previously tested higher cell densities for ffas and have noted that cell concentrations higher than 160 3 × 104 cells/well results in an overly confluent monolayer with more cells than can adhere to the 161 wells, leading to highly variable titer information (data not shown). the results of these studies 162 suggest that vero who cells plated at either 1.5 × 104 or 3 × 104 cells/well was optimal for the 163 viral ffa. 164 165 like plaque assays, the incubation time for the ffa is highly dependent on the viral 167 replication cycle within the cells and the time required for infectious progeny to be released and 168 spread to neighboring cells. whileunlike traditional plaque assaysthe ffa is not dependent 169 on viral lysis of infected cells, the development of visible spots is dependent on the time it takes 170 for viral protein production to occur and for infectious virus to spread to neighboring susceptible 171 cells. to determine the optimal time frame for infection of sars-cov-2 on a vero who cell 172 monolayer to form individual foci, we tested a variety of incubation times. in order to optimize 173 these conditions, identical dilutions of sars-cov-2 virus stocks were added to infect vero who 174 cells seeded at a density of 1.5 × 104 cells/well, and incubated for 20, 24, 48, or 72 hours post 175 infection. figure 1g shows representative images of identical dilutions of sars-cov-2 ffas 176 developed after 24, 48, or 72 hour incubation times, respectively. the mean spot size of foci at 177 each timepoint is quantified in figure 1h . the mean spot count per well at each time point is 178 quantified in figure 1i . 179 altering the incubation time had the most dramatic impact on mean foci size amid all other 180 parameters tested. while there were no significant differences in the size of foci formed between 181 20 hours post infection (hpi) and 24 hpi (p = 0.0632), we did observe the formation of 182 significantly larger foci between the 24 and 48 hpi timepoints (p = 0.0031) , as well as the 48 and 183 72 hpi timepoints (p = 0.0134) ( figure 1h) . interestingly, at the same virus dilution, we found 184 that there were fewer spots between 24 hpi (mean spot number of 10) relative to the 48 hpi time 185 point (p = 0.0369, mean spot number of 13.5) but not the 72 hpi time point (p = 0.1895, mean spot value 12.5). similarly, we found that there were no significant differences in the number of 187 foci formed between 48 and 72 hpi (p = 0.4198) ( figure 1i ). however, we noted that this 188 difference is within one standard deviation of the mean number of spots across all wells and was 189 insignificant when determining titers. from this result we concluded that incubation times greater 190 than 24 hours resulted in a slight but significant increase in spot number, while assays incubated 191 for up to 72 did not alter the spot number but did increase the spot size. this increase in spot size 192 but not number between 48 and 72 hours is a highly useful for the testing of anti-viral compounds 193 which may require longer incubation times. in addition, larger spot size makes this assay more 194 universally useful since laboratories without an automated machine can manually count spots, 195 where the large size will improve readability of the assay. 196 the ffa relies on an immunostaining protocol of an infected cell monolayer in order to 197 quantify infectious virus titer and is therefore dependent upon sars-cov-2-specific antibody 198 binding. for this purpose, polyclonal guinea pig sera (bei: nr-0361) raised against sars-cov-199 2 produces reproducible staining with minimal background. however, we have also used human as efforts to understand replication and transmission of sars-cov-2 are underway and 208 vaccine development moves forward, more information regarding permissive cell types for sars-209 cov-2 infection and replication is needed. to determine the permissivity of several different cell 210 types to infection with sars-cov-2, we generated multistep growth curves for human, non-211 human primate, murine, hamster, and gastric adenocarcinoma cell lines. each of these cell lines 212 was infected at a moi = 0.05 and cells or cell supernatants were collected aseptically, and total 213 rna was isolated. cellular rna was normalized to an internal rnasep control for human and 214 non-human primate cells and gapdh for murine cells. genome equivalents were determined 215 using the applied biosystems taqman gene expression assay protocol for sars-cov-2 216 previously described [34] . 217 to identify susceptible cell lines, we first assessed genome copy number in non-human 218 primate african green monkey kidney epithelial cells (vero who, vero e6) as well as human 219 hepatocytes (huh7.5, huh7) and lung epithelial cells (a549, calu-3). figure 2a shows the 220 sars-cov-2 genome copy number for whole cells for human and non-human primate cell lines. 221 figure 2b shows the sars-cov-2 genome copy number for cell culture supernatants for human 222 and non-human primate cell lines. in each cell line aside from vero who and huh7, genome 223 equivalents within the cell peaked at 24 hpi and remained relatively constant for the duration of 224 the experiment. sars-cov-2 genome equivalents in vero who and huh7 cells peaked at 48 hpi 225 and remained relatively constant for the duration of the experiment. the vero e6 cell line reached 226 the highest titer at 6.44 × 108 copies/µl at 24 hpi, with the huh7.5 and calu-3 cell lines reaching 227 1.1 × 106 copies/µl and 5.0 × 105 copies/µl, at the same time point, respectively. vero who and 228 huh7 cell lines reached the highest titer, 1.3 × 106 copies/µl and 1.1 × 106 copies/µl, 229 respectively, at 48 hpi. the a549 cells, although susceptible to infection, appeared to support 230 little sars-cov-2 replication, reaching only 80 copies/µl at 24 hpi. 231 we also measured genome copy number in the cell culture supernatant for all of the cell 232 lines described ( figure 2b ) for each cell line, viral rna in the supernatant peaked at later 233 timepoints, either 72 hpi (vero who, 4.7× 104 copies/µl; vero e6, 1.4× 104 copies/µl; calu-234 3, 1.3× 104 copies/µl) or 96 hpi (a549, 2.6 copies/µl; huh7, 27 copies/µl; huh7.5, 8.6 × 103 235 copies/µl). of these cell lines, the vero who cells had the highest titers in the supernatant, while 236 vero e6, huh7.5 and calu-3 cells were comparable in terms of titer. as expected, a549 cells 237 that did not contain high titers of cell-associated virus also did not contain high titers in the 238 supernatant. interestingly, while huh7 cells support relatively high titers of cell-associated virus, 239 they do not appear to yield high titers in the supernatant. these results suggest that vero e6 cells 240 are most permissible for sars-cov-2 replication among all tested cell types and would be the 241 ideal choice for propagation. vero who, huh7.5, and calu-3 cells are also permissible cell 242 types for sars-cov-2 infection and replication, however a549 cells do not appear to be suitable 243 for high levels of sars-cov-2 replication. our results also suggest that huh7 cells appear to be 244 permissible for sars-cov-2 infection and replication, but do not appear to be suitable for egress 245 into the cell culture supernatant. 246 due to ongoing sars-cov-2 vaccine development efforts, there is an urgent need to 247 develop and evaluate the susceptibility of small animal models to sars-cov-2 infection and covid-19. recent studies have suggested that rodents may be used for these purposes, as well as 249 to study the adaptive immune response to sars-cov-2 infection [34] [35] [36] [37] . to this end, we next 250 sought to determine permissivity of rodent cell lines to sars-cov-2 infection, namely 3t3 and 251 shhc17 cell lines. figure 2c shows the sars-cov-2 genome copy number for whole cells for 252 murine and hamster cell lines, with vero who cells included for comparison. figure 2d shows 253 the sars-cov-2 genome copy numbers for cell culture supernatants derived from murine and 254 hamster cell lines, with vero who supernatant included for comparison. from total cellular rna, 255 we detected only 42 copies/µl in 3t3 cells at 72 hpi, the time point at which the titer peaked. 256 similarly, with shhc17 cells, we detected only 50 copies/µl at 24 hpi, the time point at which 257 the titer peaked. in addition to total cellular rna, we also examined supernatants from cell culture 258 and predictably found peak titers of only 3 copies/µl in 3t3 cells and just 33 copies/µl in 259 shhc17 cells, both at 96 hpi. these results suggest that neither 3t3 nor shhc17 cell lines are 260 suitable for supporting sars-cov-2 replication or egress without further experimental 261 finally, because it is known that hace2 is highly expressed by intestinal epithelial cells, 263 we sought to examine the permissivity of human gastric adenocarcinoma cell lines to sars-cov-264 2 infection [15] . for this purpose, we used ags and mkn cell lines, and examined viral genome 265 copies associated with both total cellular rna as well as the cell supernatant. figure 2e shows 266 the sars-cov-2 genome copy numbers for whole cells from gastric adenocarcinoma lines, with 267 vero who cells included for comparison. figure 2f shows the sars-cov-2 genome copy 268 numbers for cell culture supernatants from gastric adenocarcinoma lines, with vero who 269 supernatant included for comparison. we found that mkn cells yielded relatively high titers, with 270 cell-associated virus peaking at 1.0× 106 copies/µl at 24 hpi. virus in the supernatant peaked at 271 5.0 × 104 copies/µl at 72 hpi. ags cells yielded lower titers, with cell-associated virus peaking 272 at 4.6× 103 copies/µl at 24 hpi and virus in the supernatant peaking at 6.7 × 102 copies/µl 96 273 hpi. interestingly, however, the titer in ags cells appeared more variable compared to other time 274 points, increasing at 48 and 96 hpi and dropping at 24 and 72 hpi. these results suggest that these 275 gastric adenocarcinoma cell lines can support infection, replication and egress of sars-cov-2 as 276 well as, or in some cases better than, vero cell lines. 277 given that our studies conducted to quantify sars-cov-2 viral genome copies in 280 susceptible cell lines yielded results that highlighted highly permissive cell lines, like vero who, 281 while also distinguishing less permissive cell lines, like a549, we sought to analyze spike and 282 hace2 protein co-expression to determine if higher hace2 expression correlated with higher 283 susceptibility to sars-cov-2 infection. 284 one facet of our understanding of the current sars-cov-2 outbreak that is rapidly 287 evolving is sars-cov-2 seroprevalence in the general population. at the same time, forming a 288 better understanding of and ability to assess the kinetics of the neutralizing antibody response to 289 sars-cov-2 could be essential in further vaccine and anti-viral development efforts. to this end, 290 we adapted the sars-cov-2 ffa for the quantification of neutralizing antibody (nab) titers in 291 the form of an frnt. this was accomplished by incubating serially diluted convalescent serum 292 from sars-cov-2 infected individuals with a known quantity of infectious sars-cov-2 (~60 293 ffu) and measuring foci formation. infection was normalized to a pbs control to reflect the 294 percent neutralization of sera. 295 first, we sought to determine whether the frnt could be used to detect a range of nab 296 concentrations in human samples. figure 4a shows the neutralization curves for human sera 297 samples, showing a decrease in virus neutralization as the serum is diluted out. these samples 298 were collected from 4 human subjects at the university of puerto rico following a positive qpcr 299 test for sars-cov-2. all subjects were in the convalescence period at the time of sample 300 collection. these assays were performed using deidentified residual sera samples. 301 using the frnt approach, we quantified the neutralizing antibody titer in the form of the 302 reciprocal serum dilution required to neutralize 50% of virus, or the frnt50 value. this assay can 303 also be used to determine the frnt90 value (i.e. required for 90% neutralization). the frnt50 304 and frnt90 values are reported in figure 4b . the reciprocal serum dilutions required for 50% 305 neutralization for the human samples (hs_a, hs_c and hs_d) are 2.161, 3.183, and 2.002, 306 respectively. the reciprocal serum dilutions required for 90% neutralization for hs_a, hs_c and 307 hs_d are 1.377, 2.725, and 1.739, respectively. for hs_b, the reciprocal serum dilution required 308 to neutralize both 50% and 90% of the virus was below the lower limit of quantitation (lloq) for 309 the assay. in order to increase confidence that these nabs were the result of recent sars-cov-2 311 infection rather than cross-reactivity with the four circulating human common cold coronaviruses, 312 we performed an elisa to examine binding of these sera samples to sars-cov-2 receptor-313 binding domain (rbd). figure 4c shows the absorbance at 450 nm (a450) values indicating that 314 sera from these subjects contain antibodies that can bind specifically to the receptor binding 315 domain (rbd) of sars-cov-2. 316 having confirmed that our assay can detect nab to sars-cov-2 in human sera, we next 317 sought to demonstrate that this assay is applicable for numerous sera sources including non-human 318 primates and mice. to this end, we performed an frnt with non-human primate (nhp) sera, 319 which consisted of pooled sera samples from a group of rhesus macaques in the convalescent 320 phase following sars-cov-2 infection by multiple routes (bei: nr-52401). figure 4e shows 321 the neutralization curve for this pool of nhp sera. low but detectable nab titers were present in 322 this sample with an frnt50 value of 2.161 and frnt90 of 1.377, as depicted in figure 4f . 323 having shown that our assay can be utilized to quantify nabs in nhp samples, we next 324 sought to demonstrate that this assay could also be used for quantifying nabs in small animal 325 models such as mice. this also afforded us the opportunity to examine the nab response both at 326 having demonstrated that our frnt assay can be used to quantify nab titers for human 335 samples, non-human primates, and mice both in acute infection and memory responses, we next 336 sought to determine whether this assay could be used to quantify nab resulting from a subunit or 337 dna vaccine. to this end, we immunized c57bl/6 mice intramuscularly (i.m) with 50 µg of 338 dna encoding the sars-cov-2 spike (ms_c). a subset of these mice was boosted 21 days later 339 (ms_b, ms_d) with 5 µg of dna intramuscularly and sera collected 21 days following the boost. 340 figure 4g shows the neutralization curves for these mice immunized with dna encoding the immunization. from these data we were able to define the frnt50 values for ms_b, ms_c, and 343 ms_d, which are shown in figure 4h , and are 2.045, 1.584, 1.227, respectively. however, the 344 frnt90 values were below the lloq for the assay, as well as the frnt50 value for ms_a. these 345 results suggest that the frnt can be used to detect nabs in sera resulting from immunizations, in 346 addition to nabs in human, nhp, and mouse sera resulting from sars-cov-2 infections. 347 348 to address the need for high-throughput, rapid quantification of infectious sars-cov-2, 350 our group has developed a focus forming assay (ffa) for sars-cov-2 using vero who cells. 351 the strength of the ffa is the rapid visualization of individual foci forming from a single 352 infectious unit or focus forming unit (ffu). the ffa for sars-cov-2 can be developed in as 353 little as 24 hours, shorter relative to traditional plaque assays for human coronaviruses which can 354 take 2-5 days [32, 38, 39] . the focus forming assay is also amenable to a 96-well plate format, 355 allowing for assays to be scaled up or automated to handle large volumes of samples quickly 356 relative to assays requiring plates with 24 wells or fewer. automating the quantification of foci 357 using equipment such as a ctl machine can also streamline the process of screening large 358 numbers of samples. one potential disadvantage of the focus forming assay is the requirement of 359 a sars-cov-2 specific antibody as the primary antibody for foci immunostaining. however, for 360 our assays we have found that polyclonal guinea pig serum provides reproducible staining with 361 minimal background when used at the appropriate concentrations, and numerous human 362 monoclonal antibodies are now commercially available and suitable for this purpose [40] [41] [42] . 363 in regard to the focus forming assay development, we initially hypothesized that the 364 absence of in vero e6 cells would make them more susceptible to sars-cov-2 infection and 365 therefore a more sensitive choice of cell line for the focus forming assay. surprisingly, we found 366 that vero who cells were more suited to foci formation. it is worth noting that other labs have 367 shown that a higher titer and larger, clearer plaques result when vero e6 cells are used in place of 368 vero who cells when performing plaque-assay based titrations with sars-cov-2 [32]. this may 369 reflect differences between the wuhan clinical isolate used in this study as opposed to the usa-370 wa1/2020 isolate or this may be an artifact of the focus forming assay. because we find that by 371 qpcr, genome copy number is typically highest in vero e6 cells, we hypothesize that more defective or non-infectious virus results from replication in vero e6 cells. additionally, high levels 373 of genome replication in vero e6 may not correlate with ability to spread laterally in cell culture 374 and form foci. the discrepancy in sars-cov-2 replication in these two cell lines warrants further 375 study. 376 our understanding of the impact of cell type on sars-cov-2 entry, replication, assembly, 377 and egress is in its infancy. these gaps in our knowledge were recently made evident by the use 378 of chloroquine and hydroxychloroquine-widely used anti-malarial drugs that create suboptimal to advance our understanding of the sars-cov-2 life cycle in susceptible cell types, we 390 generated multi-step growth curves for a variety of human, simian, and rodent cell types. in most 391 cases, viral replication peaked at 24 hpi in susceptible cell lines and this cell-associated virus was 392 maintained for the duration of the experiment. in many cases, however, the presence of virus in 393 the supernatant did not peak until 72-96 hpi. in the context of the viral replication cycle, our data 394 suggests that genome replication in vitro peaks after just 24 hours, however assembly and egress 395 from infected cells may take as long as 72-96 hpi. 396 while there are conflicting reports concerning the suitability of huh7 cells for sars-cov-397 2 studies [32, 33] we observed a striking discrepancy was between cell-associated virus within the 398 total rna and the virus detected within the cell supernatant. as much as 1.1 × 106 copies/µl of 399 cell-associated virus was detectable in rna isolated from huh7 cells, but virtually no detectable 400 virus was found in the cell supernatant. this suggested to us that viral entry-and hence the 401 production of cell-associated virus within the total rna fraction-was independent of successful 402 viral egress. this trend did not hold for rig-i-deficient huh 7.5 cells [49] , suggesting that viral egress is interferon (ifn) sensitive. this observation is in accordance with previous studies 404 describing sars-cov-2 sensitivity to type i ifns [50, 51] . further studies are warranted to 405 determine what factors are necessary and sufficient for viral egress and could therefore serve as 406 potential therapeutic targets. 407 cov-2 infection, pathogenesis, and possibly transmission [35, [52] [53] [54] , which may reflect differing 409 susceptibility of hamster cell types based on anatomical location of the isolated cells. 410 we did not observe a strong correlation between ace2 and viral spike protein levels, nor 411 did we see a strong relationship between viral genome copy and ace2 mrna level. our results 412 suggest that host cell susceptibility to sars-cov-2 infection is more complicated than ace2 413 expression alone, thus warranting further investigation. 414 we have showed by elisa that convalescent sera sourced from human, non-human 416 primate, and mice infected with sars-cov-2 can bind to the rbd of sars-cov-2. while the 417 s2 subunit of the sars-cov-2 spike protein is highly conserved among betacoronaviruses, 418 previous studies have showed that the rbd within the spike protein of sars-cov-2 is unique 419 [16, 55] . in serological studies of sars-cov-2, the presence of antibodies binding sars-cov-2 420 rbd is considered the most sensitive and specific indicator of previous sars-cov-2 exposure. 421 these results increase our confidence that within these polyclonal sera samples are neutralizing 422 antibodies that are specific to sars-cov-2, rather than cross-reactivity due prior coronavirus 423 exposure, as has been called into question by some [28, 55, 56] . 424 as other labs have noted [57] we observed that binding to sars-cov-2 rbd appears to 425 correlate with neutralization capacity, as human samples with a high auc for rbd binding by 426 elisa also had lower ec50 values, indicating that low concentrations of sera from these patients 427 were sufficient to neutralize 50% of a standardized amount of virus. we have showed that each of 428 these samples can effectively neutralize sars-cov-2 in vitro and that neutralization can be 429 with 100µl per well of diluted samples. this plate containing sample dilution on the cell 466 monolayer was placed in an incubator with 37°c, 5% co2 for 1 hour. a solution of 2% 467 methylcellulose (sigma-m0512-250g) in 1 × pbs was made in advance of the assay and stored at 468 4°c until ready to use. on the day of the assay and during the one-hour infection period, 2% 469 methylcellulose was diluted 1:1 in 5% dmem and placed on a rocker to mix. the 1% 470 methylcellulose-media mixture (hereby referred to as overlay media) was stored at room 471 temperature until ready to use. after the one-hour infection period, the 96 well plate containing 472 sample dilution and cell monolayer was removed from incubator. overlay was added to the plate 473 by adding 125 µl of overlay media to each well. this step reduces the uncontrolled spread of virus 474 throughout the monolayer on the well, making it difficult to distinguish individual foci. after the 475 addition of overlay media, the plate was returned to an incubator with 37°c, 5% co2 for 24 hours. the plate was removed from the incubator and the media containing the overlay and sample was 481 aspirated off. one wash with 150µl of 1 × pbs per well was performed, taking care not to disrupt 482 the cell monolayer. 50µl per well of 5% pfa in pbs was added for the fixing step. with the 5% 483 pfa still in the plate, the plate was submerged in a bath of 5% formalin buffered phosphate 484 (fisher: sf100-4) in 1 × pbs for 15 minutes at room temperature. after 15 minutes, the plate was 485 removed from the formaldehyde bath and the 5% pfa removed from the monolayer. one wash 486 with 100µl of 1 × pbs (tissue culture grade) per well was performed. the plate was submerged 487 in a bath of 1 × pbs to rinse and removed from bsl-3 containment. foci were visualized by an 488 immunostaining protocol. the 96-well plate was first washed twice with 150µl per well of ffa 489 wash buffer (1 × pbs, 0.05% triton x-100). the primary antibody consisted of polyclonal anti-490 sars-cov-2 guinea pig sera (bei: nr-0361) and was diluted 1:15,000 with ffa staining buffer 491 (1 × pbs, 1mg/ml saponin (sigma: 47036)).then, 50µl per well of primary antibody was allowed 492 to incubate for 2 hours at room temperature or 4°c overnight. the 96-well plate was then washed 493 three times with 150µl per well of ffa wash buffer. the secondary antibody consisted of goat 494 anti-mouse conjugated horseradish peroxidase (sigma: a-7289) diluted 1:5,000 in ffa staining 495 buffer. similarly, 50µl per well of secondary antibody was allowed to incubate for 2 hours at room temperature or 4°c overnight. the plate was washed three times with 150ul per well of ffa 497 wash buffer. finally, 50µl per well kpl trueblue hrp substrate was added to each well and 498 allowed to develop in the dark for 10-15 minutes, or until blue foci are visible. the reaction was 499 then quenched by two washes with millipore water and imaged immediately thereafter with a ctl 500 machine to quantify foci. 501 502 briefly, sera samples were diluted 1:40 in 5% dmem and added to the topmost row of a round 504 bottom 96-well plate. sera samples were then serially diluted 1:3 down the remainder of the plate 505 in 5% dmem. an equal volume of sars-cov-2 diluted to ~600 ffu/ml (~60 ffu/100µl) was 506 then added to the serially diluted sample, mixed thoroughly, and allowed to incubate at 37°c for 507 1 hour. then 100µl of sars-cov-2+sera mixture was transferred to a vero who cell monolayer 508 (as described in the focus forming assay). from this point, the assay was as described in the focus 509 forming assay section. 510 511 binding of human polyclonal sera to recombinant sars-cov2 proteins was determined by 513 elisa. a 1ug/ml mixture of 50µl per well containing recombinant protein in carbonate buffer 514 (0.1m na2co3 0.1m nahco3 ph 9.3) was used to coat maxisorp (thermofisher) 96-well plates 515 overnight at 4°c. plates were blocked with blocking buffer (pbs + 5%bsa + 0.5% tween) for 2 516 hours at room temperature the following day and washed four times with wash buffer. polyclonal 517 sera was serially diluted in blocking buffer prior to plating. sera was allowed to incubate for 1 518 hour at room temperature and washed four times with wash buffer. following the one hour 519 incubation, goat-anti-human igg hrp (sigma) conjugated secondary (1:5000) was added and 520 allowed to incubate for 1 hour at room temperature. the plate was washed again four times with 521 wash buffer and the elisa was visualized with 100µl per well of tmb enhanced substrate 522 (neogen diagnostics) and allowed to develop in the dark for 15 minutes. a solution of 1n hcl 523 was used to quench the reaction and the plate was read for an optical density of 450 nanometers 524 on a biotek epoch plate reader. the total peak area under the curve (auc) was calculated using 525 graphpad prism 8. 526 isolation of rna from cell culture and culture supernatants 528 rna was isolated from cell culture and supernatant using an invitrogen purelink rna mini kit 529 according to the manufacturer's instructions. 530 531 rt-qpcr 532 hace2 expression was measured by qrt-pcr using taqman primer and probe sets from idt 533 (assay id hs.pt.58.27645939). sars-cov-2 viral burden was measured by qrt-pcr using 534 taqman primer and probe sets from idt with the following sequences: were allowed to infect cell monolayer for 1 hour at 37°c, 5% co2 before overlay with 2% 566 methylcellulose in 5% dmem. following a 24 hour incubation at 37°c, 5% co2, cells were fixed 567 in a solution of 5% paraformaldehyde diluted in 1 × pbs. foci were visualized via immunostaining 568 with polyclonal guinea pig anti-sars-cov-2 sera and goat anti-guinea pig conjugated hrp. kpl neutralization capacity was measured by mixing serially diluted sera with a standardized amount 614 of virus (~60 ffu) and allowed to incubate for 1 hour at 37°c, 5% co2 before allowed to infect a 615 cell monolayer as described in figure 1 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 624 medicine early transmission dynamics in wuhan, china, of novel coronavirus-infected 626 pneumonia identification of a novel coronavirus causing severe pneumonia in 628 human: a descriptive study a new coronavirus associated with 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specific cell subsets across tissues specific ace2 expression in small intestinal enterocytes may cause 655 gastrointestinal symptoms and injury after 2019-ncov infection structure, function, and antigenicity of the sars-cov-2 spike 659 glycoprotein tmprss2 and tmprss4 promote sars-cov-2 infection of human small 661 intestinal enterocytes syndrome coronavirus spike protein for membrane fusion and reduces viral control by 664 the humoral immune response neutralizing antibodies correlate with protection from sars-cov-2 in 666 humans during a fishery vessel outbreak with high attack rate. medrxiv neutralizing antibody responses to sars-cov-2 in a covid-19 recovered 669 patient cohort and their implications. medrxiv, 2020: p. 2020.03.30 quantifying antibody kinetics and rna shedding during early-phase 671 sars-cov-2 infection antibody responses to sars-cov-2 in patients with covid-19 the epidemiology and clinical information about covid-19. european 675 journal of clinical microbiology & infectious diseases : official publication of the european 676 society of clinical microbiology clinical characteristics of coronavirus disease 2019 in china the epidemiology and pathogenesis of coronavirus 680 disease (covid-19) outbreak sars-cov-2 and viral sepsis: observations and hypotheses. the lancet clinical findings in a group of patients infected with the 2019 novel 684 coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series the receptor-binding domain of the viral spike protein is an 687 immunodominant and highly specific target of antibodies in sars-cov-2 patients propagation, quantification, detection, and 690 storage of west nile virus development and optimization of a direct plaque assay for human and 693 avian metapneumoviruses enhanced isolation of sars-cov-2 by tmprss2-expressing cells isolation and characterization of sars-cov-2 from the first us 697 covid-19 patient sars-coronavirus-2 replication in vero e6 cells: replication kinetics, 699 rapid adaptation and cytopathology mrna induced expression of human angiotensin-converting enzyme 2 701 in mice for the study of the adaptive immune response to severe acute respiratory syndrome 702 coronavirus 2. biorxiv syrian hamsters as a small animal model for sars-cov-2 infection and 704 countermeasure development mouse model of sars-cov-2 reveals inflammatory role of 707 type i interferon signaling. biorxiv a sars-cov-2 infection model in mice demonstrates protection by 709 neutralizing antibodies plaque assay for human coronavirus nl63 using 711 human colon carcinoma cells two detailed plaque assay protocols for the quantification of 713 infectious sars-cov-2. current protocols in microbiology potently neutralizing and protective human antibodies against sars-cov-716 2 potently neutralizing human antibodies that block sars-cov-2 receptor 718 binding and protect animals. biorxiv, 2020: p. 2020.05.22.111005 rapid isolation and profiling of a diverse panel of human monoclonal 720 antibodies targeting the sars-cov-2 spike protein hydroxychloroquine, a less toxic derivative of chloroquine, is effective in 722 inhibiting sars-cov-2 infection in vitro remdesivir and chloroquine effectively inhibit the recently emerged novel 724 coronavirus (2019-ncov) in vitro in vitro antiviral activity and projection of optimized dosing design of 726 hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 727 2 (sars-cov-2) a randomized trial of hydroxychloroquine as postexposure 729 prophylaxis for covid-19 chloroquine does not inhibit infection of human lung cells with sars-731 cov-2 proteolytic processing of middle east respiratory syndrome coronavirus 733 spikes expands virus tropism regulating intracellular antiviral defense and permissiveness to 736 hepatitis c virus rna replication through a cellular rna helicase, rig-i sars-cov-2 is sensitive to type i interferon pretreatment. 739 biorxiv : the preprint server for biology antiviral activities of type i interferons to sars-cov-2 infection. 741 antiviral research pathogenesis and transmission of sars-cov-2 in golden hamsters massive transient damage of the olfactory epithelium associated with 745 infection of sustentacular cells by sars-cov-2 in golden syrian hamsters. brain, 746 behavior, and immunity surgical mask partition reduces the risk of non-contact transmission 748 in a golden syrian hamster model for coronavirus disease 2019 (covid-19) a serological assay to detect sars-cov-2 seroconversion in humans cross-reactivity towards sars-cov-2: the potential role of low-pathogenic 753 human coronaviruses. the lancet microbe human neutralizing antibodies elicited by sars-cov-2 infection key: cord-339012-4juhmjaj 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: 2020-07-28 journal: biorxiv doi: 10.1101/2020.07.28.224576 sha: doc_id: 339012 cord_uid: 4juhmjaj the transcriptional response in vero cells (atcc® ccl-81) infected with the coronavirus porcine epidemic diarrhea virus (pedv) was measured by rnaseq analysis 4 and 6 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. il6, cxcl10, il1a, cxcl8 [alias il8]) and antiviral genes (e.g. ifi44, ifit1, mx1, 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 6h. 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 tmprss13 (alias mspl) to support its own infection by virus-cell membrane fusion (shi et al, 2017, viruses, 9(5):114). 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 il1b, tnf, cxcl8 and ptgs2. the only 2 cytokine genes found up-regulated by pedv were cardiotrophin-1, an il6-type cytokine with pleiotropic functions on different tissues and types of cells, and endothelin 2, 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-2. this may help them to fine-tune therapeutic treatments and apply specific approved drugs to treat covid-19 patients. the lack of knowledge for treating hospitalized sars-cov-2 infected patients is one of the pressing problems of the current covid-19 pandemic. the sars-cov-2 virus shows a close genetic similarity to the in april 2003 identified sars virus (sars-cov-1) and to other sars-related coronaviruses isolated from humans and bats. sars-cov-2 induces clinical respiratory symptoms familiar to the 2003 virus, mostly in persons with underlying diseases like copd, heart failure, diabetes and obesity (1: wu et al. 2020) . despite the 2003 sars-cov-1 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-2 infections. in our lab we study the alpha-coronavirus pedv. pedv was first detected in pig herds in 1977 in europe (2: pensaert and de bouck 1978) . however, this virus reemerged in the spring of 2013 in north america causing a massive outbreak among pig herds, resulting in the death of about 30% 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 mrv3 in pedv infected piglets, also other mrv serotypes were isolated from hospitalized patients with airway problems diagnosed positive for sars-cov-1 (11: cheng et al. 2009 , 12: duan et al. 2003 , 13: zuo et al. 2003 . recently, a cross-family recombinant coronavirus was isolated in china from bat faeces in which an rna sequence originating from the s1 segment of mrv was inserted in the coronavirus genome between the n and ns7a genes, indicating that both viruses were replicating simultaneously in a single cell in bats (14: huang et al. 2016) . a prevalence study showed that this cross-family recombinant coronavirus circulated in an isolated bat colony in a cave in china (15: obameso et al. 2017 ). 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 (4 and 6 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-1 and -2. however, the genomic organization, replication strategy and function of a part of the viral nonstructural proteins share common features among all coronaviruses (16: brian and baric 2005) . 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-81) because these cells support efficient infection and replication of both mrv and pedv. vero cells are susceptible for many coronaviruses, including sars-cov-1 and -2 (17: chu et al. 2020). they originate from epithelial tissue, in part resembling nasal and bronchial epithelium cells, the prime target cells infected by sars-cov-2 in the airways of humans. recent research showed that sars-cov-2 is also able to replicate in epithelial cells of human small intestinal organoids (18: lamers et al. 2020) . a disadvantage of vero cells is a deletion in the type i interferon (ifn) gene cluster on chromosome 12 (19: osada et al. 2014 ). 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 3 (20: chew et al. 2009 ). single infections with pedv or mrv3 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-2 pathogenesis. because of the covid-2 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 mrv3. 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-2 and influenza host responses by rnaseq (21: blanco-melo et al. 2020). 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-19 disease. a time-dependent infection experiment was performed with cultured vero cells. details are described in supplementary file 1 (material and methods) and visually displayed in this file. briefly, overnight cultured vero cells grown in 2 cm 2 wells were mock-infected, infected with mrv3 strain wbvr (7: hulst et al. 2017) or pedv strain cv777 (2: pensaert and de bouck 1978, 22: rasmussen et al. 2018] ) with a multiplicity of infection of ≥1 for 30 min at 4°c. for pedv and corresponding mockinfected cells, 10 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 30 min at 4°c, virus was discarded and cells were washed twice and supplied with fresh culture medium. cells were incubated for 0, 2, 4, 6, and 16h at 37°c and 5% co2. 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.1 : methods and primers used for pcr are provided in supplementary file 1). in addition, cells in one of the quadruplicate wells incubated for 16h were fixated and stained with antibodies directed against the s2 spike protein of pedv and the s1 attachment protein (α1) of mrv3. a decrease in ct-values for pedv was not observed before 6 h post inoculation (6 h.p.i), indicating that replication in pedv infected cells started later than was observed for mrv (at 4 h.p.i). staining of the cells after 16h indicated that nearly all vero cells were infected with mrv3 and more than 50% with pedv. also more than 50% of the cells in 16hwells appeared as fused cells (syncytia), confirming that more than 50% 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 16 h.p.i. were partially degraded (rin values below 9), making them unsuitable for rnaseq analysis. therefore, only 0, 4 and 6h timepoints were analyzed using rnaseq. stained with a monoclonal antibody directed against the s2 spike protein. mrv and mock infected cells were stained with a polyclonal rabbit serum raised against a peptide sequence of the s1-attachment protein of mrv serotype 3. nuclei were stained blue with the hoechst, 4',6-diamidino-2-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 2a 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 0, 4, and 6h by genomescan (see supplementary file 2b). from these datafiles we extracted lists of degs with a fc>2 and p-value of <0.05. 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 3 sheet "pedv-mrv degs fc>2", lists of all annotated pedv and mrv degs are presented with their fc. in a separate sheet "pedv-degs functional info" all 266 individual degs regulated by pedv at 4 and 6 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 3 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 3). 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 <0.05) were retrieved and listed in 3 separate sheets in supplementary file 3 (sheets "mrv-pedv pathways", "pedv g0-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 1 . 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 1 (see sheet "mrv-pedv pathways" in supplementary file 3 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-2 (explained below) using the genotyping program varelect (lifemap sciences, inc.) and displayed in supplementary file 3 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 >10 (up) or <-10 (down) or playing an important role in biological processes induced by pedv and related to covid-19 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-2 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 1 . 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 2 matching genes were retrieved from geneanalytics. common pathways for mrv were included in table 1a . a full list of pathways with degs, retrieved for mrv at 4 and 6h, is provided in supplementary file 3 (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 4 and 6h by pedv. in fig. 2a 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 cxcl10 and cxcl8 (alias il8), already at 4 h.p.i., whereas pedv did not, even not when replication of pedv rna was detected by rt-qpcr at 6 h.p.i.. for mrv, this cytokine/chemokine response at 4 h.p.i. was followed by high up-regulation of "hallmark" antiviral genes at 6 h.p.i. (see fig. 2b : e.g. interferoninduced genes [ifi] and oasl) and chemokines that attract t cells, monocytes, granulocytes, including basophils (e.g. cxcl8, cxcl11 and ccl2). pedv infection up-regulated only a few genes coding for proteins with cytokine activity (ctf1 and edn2), and also did not elevated gene expression of these "hallmark" antiviral genes. in contrast, pedv down-regulated expression of 6 genes (out of 9 in total) coding for zinc finger proteins (out of 9 in total), all 6 with an antiviral activity towards herpes simplex virus 1 ( fig 2c) binding of thrombin to f2rl2 reduces inflammation, activates platelets and increases vasodilation and permeability of the vascular wall (see also below in the section "platelets activation"). csf1r is a receptor for the cytokine colony stimulating factor 1, 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 3, 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. 3, aoc1 , maoa, il4i1). 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 3, sheet "biogenic amines"). three enzymes clustered in the "histidine metabolism" pathway (https://www.kegg.jp/keggbin/show_pathway?hsa00340+4128) with histamine and reaction products generated from this biogenic amine (fig. 3) . also most association of pedv-degs were found by varelect for histamine. the gene coding for the amine oxidase "interleukin 4 induced 1" (il4i1) was strongly down-regulated (30-fold) 4h after infection with pedv. besides catalysis of l-phenylaniline into phenylpyruvate (fig.3) , il4i1 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 2 (ptgs2) gene was upregulated by mrv at 6h p.i. ptgs2 synthesizes prostaglandin endoperoxide h2 (pgh2), an compound with a short half-life and the precursor of many biological active prostaglandins: e.g. thromboxane-a2 (mediates activation of platelets), pgi2 and pge2. in contrast, pedv increased the expression of the gene coding for prostaglandin e synthase (ptges) which converts pgh2 into pge2. pge2 is a direct vasodilator, but does not inhibit platelet aggregation. pge2 also suppresses t cell receptor signaling. pedv decreased expression of the gamma-glutamyltransferase 1 gene (ggt1) after 4h (2-fold), but increased expression of this gene 2 hours later to a 4-fold level compared to mock infected cells. ggt synthesizes leukotriene d4 (ltd4) from ltc4. ige-activated mast cells may secrete ltd4 and ltc4, 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 1 ). 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. 4 ), 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 3, 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 (rnd1 and sema5a), ii) inhibit formation of cilia (kinases mak1 and cdk20, highly up-regulated at 6h), and iii) regulate cytoskeleton rearrangements that facilitate axon growth and growth and stabilization of dendritic spines (f2rl2, 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 4) . in particular, the highly down-regulated gene il4i1 (30-fold at 4h p.i.) is of interest (see also above). il4i1 is believed to be secreted from apc's (e.g. dc's) in the immune cleft formed with t cells (31: molinier-frenkel et al. 2019). the mechanism how il4i1 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 h2o2 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 il4i1, amino oxidase maoa (down-regulated 11-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 2 subfamily a member 14 (or2a14; 17-fold at 4h) and anoctamin 2 (ano2, alias cacc;14-fold at 4h). ano2 is a calcium-activated chloride channel imbedded in the basal membranes of neurons that harbor apical membrane receptors like or2a14 that sense odorants. by importing chloride ions into the cytosol ano2 contributes to the depolarization of these neurons (https://www.kegg.jp/kegg-bin/show_pathway?hsa04740+57101). loss of smell and taste is one of the first noticeable symptoms of covid-19. genetic defects in the ano2 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 3 in separate sheets for all 4 queried terms. degs matching to more than one query term are displayed in fig. 5 . remarkable associations of degs with these terms are mentioned in sections beneath. . edn2 and agt2 are vasoactive peptides and binding of edn2 and agt2 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?hsa04924+1907). ren converts pre-angiotensinogen (agt) to the endocrine peptide-hormone agt1 (https://www.kegg.jp/kegg-bin/show_pathway?hsa04614+5972). agt1 is further cleaved to variants with specific endocrine activity by angiotensin i converting enzymes (e.g. ace and ace2: ). on the surface of bronchial epithelial cells ace2 was identified as entry receptor for sars-cov-1 and -2 (43: hoffmann et al. 2020). the octamer peptide agt2 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 (44: agapitov et al. 2002) . all processes that are important for proper functioning of the vascular system, heart muscles and kidneys. ctf1 is directly involved in the pathology of numerous cardiovascular diseases by promoting cardiac myocyte hypertrophy (41: wollert et al. 1996) , 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 4 (scn4b) was strongly upregulated (15-fold) and mylk2 (see above), citrate synthase (cs; down-regulation 24-fold) and ankyrin repeat domain 1 (ankrd1) were strongly down-regulated. down-regulation of cs may reduce oxidative capacity in cardiomyocytes. gene expression of ankrd1 was down-regulated 12-fold in response to mrv infection at 4 h.p.i, but reverted to a 24-fold up-regulation 2h later. ankrd1 is a putative transcription factor involved in regulation of gene expression in hypertrophic myocytes (https://www.wikipathways.org/index.php/pathway:wp516) regulation of cytosolic calcium levels in the cytosol of cardiomyocytes, e.g. by binding of col1a1 and col2a (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 cacna1h; down-regulated 5-fold at 4h by pedv) may also trigger myocyte hypertrophy. pedv strongly down-regulated gene expression of a potassium voltage-gated channel (kcnq4;16-fold). this in contrast to a strong up-regulation of the sodium symporter scn4b. for the potassium channel cacna1h (alias kv7.4) it was reported that this channel regulates the membrane potential and ca 2+ permeability of mitochondria located in the vicinity the sarcoplasmic reticulum in rat cardiomyocytes (45: testai et al. 2016). 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 3 to 4-fold for the potassium (kcnq4) and calcium channel cacna1h, but did not increased expression of the sodium symporter gene scn4b or orthologs of this gene. chronic hypertension and heart disease/failure is a complication frequently observed in obese/diabetic patients. in accordance with this, 16 out of the 65 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 2 . 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 3, sheet "pedv key degs". table 2 . 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-19 pathology. because of the differences in genomic organization and expression of viral proteins between sars-cov-2 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 (>50%) with pedv and mrv synchronically. this resulted in a unique set of highly up-and down-regulated degs for pedv. not more than 14% of the 266 pedv-degs (n=37) were similar to mrv-degs (total of 727 mrv-degs). in contrast to mrv, we observed no typical response of antiviral genes and related cytokine/chemokine genes in vero cells within 6 h.p.i. for pedv. for mrv these processes started already before 4 h.p.i.. we have to notice that pedv replication started 2h later than mrv3 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 6h 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 6h 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-1 and -2, we observed a high up-regulation of the transmembrane serine protease gene (tmprss13) that acts as a co-factor in the infection process of cells ( we observed a reduced expression of eef1a1, 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 cxcl8 (alias il8) an cxcl10. this reduced expression of eef1a1 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 mrv3 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 17 (th17) differentiation/activation" and "il17 signaling". antigen presentation by m cells to th17 cells in the submucosal layers stimulate secretion of different types of il17 cytokines (il17a-d, il25 and il17f) resulting in activation of different types of innate immune cells and t cells, including th1/th2 cells. dysregulation of this pathogen-induced il17 response may disturb the balance between th1/th2-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 il17 expression and secretion by th17 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 il17 secretion by th17 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-2 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 10-fold up-regulation of the enzyme aoc1 suggests that histamine is converted to imidazole-acetaldehyde (see fig. 3 ). 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-2 infection to release pro-inflammatory cytokines (il1, il6 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-19 patients, as part of an sars-cov-2 immune-evading strategy, and the role of il4i1 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 20-fold up-regulation of fez1 gene expression at 4h (20-fold) descended within 2h to a moderate 6-fold upregulation. this descend occurred simultaneously with elevation of gene expression of the kinases mak and cdk20, 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-2 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-2 entry receptor ace2 and its co-receptor tmprss2 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-19 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-2. 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-19 patients, or research that provides new insights for treatments with alternative prophylactic and therapeutic approved drugs. in table 3 . we summarized the pedv-degs that are, to our opinion, of interest for modulation of the biological processes underlying the pathogenesis of covid-19. table 3 . possible target genes for covid-19 therapy. colophon. the overwhelming amount of data published recently made it impossible for us to oversee all (novel) facts about the sars-cov-2 virus and pathology of the covid-19 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 3 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-19 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 11th annual 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its interaction with human cd26 therapies for bleomycin induced lung fibrosis through regulation of tgf-beta1 induced collagen gene expression covid-19-induced acute respiratory failure: an exacerbation of organspecific autoimmunity? medrxiv 2020.04 cardiotrophin-1 activates a distinct form of cardiac muscle cell hypertrophy. assembly of sarcomeric units in series via gp130/leukemia inhibitory factor receptor-dependent pathways a985g polymorphism of the endothelin-2 gene and atrial fibrillation in patients with hypertrophic cardiomyopathy sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor role of endothelin in cardiovascular disease expression and function of kv7.4 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 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subpopulations with distinct t cellsuppressive activity sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes sars-cov-2 receptor ace2 and tmprss2 are primarily expressed in bronchial transient secretory cells mechanisms of innate immune evasion in re-emerging rna viruses supplementary file 3: 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-263178-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: 2018-05-02 journal: virus res doi: 10.1016/j.virusres.2018.04.013 sha: doc_id: 263178 cord_uid: lvxxdvas to study the roles of hypervariable regions (hvrs) in receptor-binding subunit s1 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/091022 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, 2003) . 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, 2003) . the main structural protein is the spike (s) glycoprotein, which comprises a divergent s1 subunit and conserved s2 subunit (de groot et al., 1987; shil et al., 2011) . the s1 subunit contains the receptor-binding domain (wickramasinghe et al., 2011) , and it carries virus-neutralizing and serotype-specific determinants. the s1 domain exhibits high sequence diversity, where 20%-25% (even up to 50%) of the amino acids differ within the s1 subunit among ibv serotypes. after comparing the s1 subunit from a number of distinct ibv isolates, a previous study defined particularly variable segments at the amino terminus of the s1 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 s1 subunit are co-located within the hvrs (cavanagh et al., 1992 (cavanagh et al., , 1988 moore et al., 1997; niesters et al., 1987) . in addition, the hvrs are possibly associated with receptor binding. it has been reported that the critical amino acids for attachment of the m41 spike overlap with a hvr in the s1 subunit (promkuntod et al., 2014) . 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/091022 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 10% fetal bovine serum (sigma-aldrich, saint louis, mo, usa), penicillin (100 units/ ml), and streptomycin (100 μg/ml) at 37°c with 5% carbon dioxide. the ck/ch/ldl/091022 strain was isolated from h120-vaccinated chickens with renal lesions in 2009 . the beaudette strain was routinely propagated in vero cells (liu et al., , 1998 tay et al., 2012) and the 50% tissue culture infection dose (tcid 50 ) of the viral stock was calculated using 96-well plates with 10-fold serial dilutions. the 50% egg infection dose (eid 50 ) of the viral stock was determined by inoculating 10-fold dilutions into groups of 9-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., 2007) . the three hvrs in the isolate ck/ ch/ldl/091022 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/091022, and subsequently ligated into the full-length ibv cdna ( fig. 1a and table 1 ). 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., 2006) . 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 100 × eid 50 was inoculated into the allantoic cavities of 9-dayold embryonated eggs, and the allantoic fluid was harvested from three eggs in each group at 12, 24, 36, 48, and 60 h post-inoculation, where the fluids from three eggs were pooled for eid 50 determination with three replicates. vero cells were infected with beaudette and recombinant ibvs, and three wells were harvested at 4, 8, 12, 16, 24, and 36 h post-infection. viral stocks were prepared by freezing/thawing the cells three times to determine the tcid 50 with three replicates for each time. ibv infected cells cultured in six-well plates were washed with phosphate-buffered saline (pbs), fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.2% triton x-100 for 10 min. if staining was performed with a monoclonal antibody 6d10 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. sq20160408). we randomly assigned 150 7-day-old spf layer chickens to 10 groups, i.e., the seven recombinant strains, beaudette, ck/ch/ldl/ 091022, and pbs groups. there were 15 spf chickens in each group. the challenge strain (10 5 × eid 50 per bird) was applied via the intranasal and ocular routes. the clinical signs were monitored in 10 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 5 days after challenge. signs were scored as 0 = absent, 1 = mild, 2 = moderate, or 3 = severe. at 5 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 5-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 1 to 5 based on their severity (i.e., normal, mild, moderate, marked, and severe) (van ginkel et al., 2015) . moreover, viral shedding was quantified in the oropharyngeal secretions from 10 infected chickens at 4, 8, 12, 16, and 20 days after challenge by real-time rt-pcr (jones et al., 2011) . the tissues collected at 5 days post-ibv challenge, i.e., tracheas, lungs, kidneys, and cecum tonsils, were also subjected to immunohistochemistry (ihc) using monoclonal antibody 6d10, as described previously (de wit et al., 2011; xu et al., 2016) . 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/ 091022 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 200 × eid 50 or tcid 50 for the ibv strains. the two-way cross-neutralization test between the beaudette and recombinant ibvs was performed in vero cells. after incubation for 1 h at 37°c, the virus-serum mixtures were cultured in 96-well microplates for 5 days. we could not detect the replication with ck/ch/ ldl/091022 in vero cells, so the neutralization tests were performed in 9-day-old spf embryonated eggs to confirm whether the serotypes of the recombinant ibvs belonged to ck/ch/ldl/091022. 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., 1950; wadey and faragher, 1981) . 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 96-well microplates (corning, usa) (10 5 cells/well). for the adsorption assay, each strain was incubated in cells at a multiplicity of infection (moi) of 1 for 1 h at 4°c with three replicates (fang et al., 2010; sun et al., 2017) . after two washes with pbs, the microplates were fixed with methanol and 1% hydrogen peroxide for 30 min, and then permeabilized with 0.2% triton x-100 for 10 min at room temperature. next, the microplates were washed three times and blocked with 8% skim milk for 30 min at room temperature. after washing three times, the microplates were incubated with the primary mouse monoclonal antibody 6d10 for 1 h at 37°c. a similar procedure was then performed where the plates were incubated for 40 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 630 nm. for the internalization assay, cells were seeded into 96-well microplates and inoculated with each strain for 1 h at 37°c. after incubation, the cells were washed twice with pbs, before treating the infected cells with citrate buffer for 1 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 24-well plates (corning) and infected with each separate strain at an moi of 1. in the adsorption and internalization stage, total rna was extracted from the cells, before storing at −80°c for the subsequent quantification of viral loads, as described previously (liu et al., 2006a; sun et al., 2014) . three replicates were analyzed for each sample. first, the stable expression levels of three candidate reference genes comprising 18s rna, β-actin, and gapdh were compared by real-time rt-pcr in vero cells infected with the beaudette strain at 4°c or 37°c for 1 h. the results were analyzed using genorm software, normfinder software, and delta ct (http://150.216. 56.64/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., 2014) . 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 200 7-day-old spf layer chickens to 10 groups, with 20 birds in each group. birds in groups 1-9 were inoculated with the seven recombinant ibvs, beaudette, and ck/ch/ldl/ 091022 (10 5 × eid 50 per bird) via intranasal and ocular routes. birds in group 10 were inoculated with pbs and treated as the negative control. half of the surviving birds in each group were randomly selected and challenged after 21 days with a 10 6 × eid 50 dose of ck/ch/ldl/ 091022 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 4, 8, 12, 16, and 20 days after challenge by real-time rt-pcr (jones et al., 2011) . the remaining chickens in each group were investigated for 15 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 < 0.05. 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 < 0.05. 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/091022 either separately or simultaneously (fig. 1a and table 1 ). 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., 2005) . after 48 h, the virus present in each medium was collected and used to inoculate 9-day-old spf embryonated eggs. to differentiate the rescued viruses from the parental viruses, viral rna was extracted from allantoic fluid and the s1 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. 1b) . no virus antigen was found in cells infected with ck/ch/ldl/091022. rt-pcr analysis of subgenomic mrna 3 and mrna 4 was performed to confirm the virus replication (fig. 1c) (fang et al., 2007) . 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 96-well plates and infected with the recombinant ibvs and two parental strains (10 5 × eid 50 ) for 1 h at 4°c or 1 h at 37°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 50 compared with that in the beaudette strain (100%). (b) vero cells were cultured in 24-well plates and infected with the recombinant ibvs and parental strains (10 6 × eid 50 ) for 1 h at 4°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 (100%). 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 < 0.05 using anova followed tukey's multiple comparison tests. s1 gene sequencing results confirmed that the heterogenous hvrs were stably maintained in the recombinant ibvs (sup fig. 1) , 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. 1c) , the growth kinetics of the recombinant ibvs were similar to those of the beaudette strain, and they all reached their peak titer at 16 h post-infection, and there was no significant difference between the titers for the recombinant ibvs and beaudette at different time points (fig. 2) . in embryonated eggs (fig. 3a) , 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/091022 was significantly higher than those of the recombinant ibvs and beaudette at 12, 36, 48, 60 h (fig. 3b ). 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. 1b) 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 4°c for 1 h (fig. 4a) . the real-time rt-pcr results were consistent with the celisa results in the adsorption assays (fig. 4b) . 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. 4c) . the real-time rt-pcr results were consistent with the celisa results from the internalization assay (fig. 4d) . in order to test the pathogenicity of the recombinant ibvs, 7-day-old spf chickens were inoculated via intranasal and ocular routes at a dose of 10 5 × eid 50 per bird (sun et al., 2011) . we found that some of the chickens only had very low levels of snicking at 6 and 7 days postinfection, and there were no obvious clinical signs in the groups treated with the recombinant ibvs and beaudette. however, 5/10 birds (50%) died during 5-9 days post-challenge with ck/ch/ldl/091022 and the morbidity rate was 100% (fig. 5a) . 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/091022 (fig. 5b) . the autopsy results showed that ck/ch/ ldl/091022 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. 2) . we also assessed the severity scores for histopathological lesions in the trachea with deciliation (fig. 6a) , goblet cells (fig. 6b) , mucosal thickness (fig. 6c) , and lymphocytes (fig. 6d) at 5 days after challenge with the ibvs (van ginkel et al., 2015) . the scores were lowest in the beaudette group and highest in the ck/ch/ ldl/091022 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/091022. finally, no ibv rna was detected in the oropharyngeal swabs from chickens inoculated with the recombinant ibvs and beaudette after 4, 8, 12, 16, and 20 days by real-time rt-pcr, where the results were considered positive when the ct value was less than 32 zhao et al., 2017) . 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., 1950) , the serotype relatedness values were calculated based on the cross-virus neutralization studies using vero cells (table 2) or embryonated chicken eggs (table 3) . viruses with an archetti and horsfall relatedness value greater than 25 were considered to be related serotypes. our results showed that the recombinant ibvs and beaudette belonged to the same serotype (table 2) , whereas the recombinant ibvs and ck/ch/ldl/091022 belonged to different serotypes (table 3 ). in general, we found that the serotype relatedness values decreased between the recombinant strain and beaudette (table 2 ) 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/ 091022 was detected in trachea (sup fig. 3a) , cecal tonsil (sup fig. 3b) , and kidney samples (sup fig. 3c ), 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. 3d) . the viral rna could be detected by real-time rt-pcr in the selected tissues from chickens infected with ck/ch/ldl/091022, with high viral loads in the kidneys. however, very low rna levels were detected in only a few (a) mortality was recorded daily for 10 randomly selected infected birds and the survival curve was drawn. (b) at 5 days after challenge, we evaluated the challenged chickens blindly and clinical signs were scored as: 0 = absent, 1 = mild, 2 = moderate, or 3 = 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 250 (2018) 104-113 trachea samples from the birds infected with the recombinant ibvs and beaudette (fig. 7) . 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/091022 (table 4 ). viral shedding was detected in some of the chickens challenged with ck/ch/ldl/091022, 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/091022 inoculated group. these results suggest that although the recombinant ibvs could fig. 6 . trachea lesion scores. at 5 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 1 to 5 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/ 091022, inoculation with the seven recombinant ibvs did confer some cross protection against ck/ch/ldl/091022 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 s1 subunit of the s protein (cavanagh and davis, 1986; ignjatovic and galli, 1994; johnson et al., 2003; song et al., 1998) . 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/091022 strains in our study. ibv beaudette is a well-known apathogenic lab strain (geilhausen et al., 1973) , which was adapted to vero cells from chicken embryos (fang et al., 2005) . the ck/ch/ldl/091022 ibv strain is an epidemic strain isolated from h120-vaccinated layers in china. this strain mainly causes gross lesions in chicken kidneys, with high morbidity and mortality rates (liu et al., 2008 (liu et al., , 2009 liu et al., 2006b) . the ck/ch/ldl/ 091022 strain can only proliferate in chicken embryos and not vero cells. in addition, the beaudette and ck/ch/ldl/091022 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/091022. all of the recombinant ibvs proliferated in vero cells (figs. 1b and figure 2) , and had the similar growth kinetics as beaudette in vero cells (fig. 2) and embryonated eggs (fig. 3b) . the accumulation of heterogenous hvrs weakened the capacity for adsorption during infection by the recombinant viruses in vitro ( fig. 4a 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 s2, 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 7-day-old spf chickens (fig. 5e) , whereas ck/ch/ldl/ 091022 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., 2003) . virus binding to host cells is the first step in tropism determination and hvrs within the s1 subunit may affect ibv binding (wickramasinghe et al., 2011) , but s2 is responsible for membrane fusion , and thus exchanging the hvrs did not change their tropism. fig. 7 . detection of ibv replication in challenged chickens. at 5 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 32 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 < 0.05 using anova followed tukey's multiple comparison tests. 8d 12d 16d 20d 4d 8d 12d 16d 20d 4d 8d 12d 16d 20d rhvr i 7/10 1/10 10/10 4/9 2/9 1/9 0/9 0/10 2/10 3/10 7/10 10/10 6/10 9/9 9/9 9/9 9/9 rhvr ii 7/10 1/10 10/10 7/9 4/9 2/9 0/9 0/10 1/10 3/10 5/10 10/10 7/10 9/9 9/9 9/9 9/9 rhvr iii 8/10 1/10 10/10 5/9 2/9 1/9 0/9 0/10 2/10 4/10 5/10 10/10 6/10 9/9 9/9 9/9 9/9 rhvr i/ii 7/10 0/10 10/10 6/10 4/10 0/10 0/10 0/10 3/10 5/10 7/10 10/10 7/10 10/10 10/10 10/10 10/10 rhvr i/iii 7/10 1/10 10/10 7/9 2/9 0/9 0/9 0/10 2/10 4/10 6/10 10/10 6/10 9/9 9/9 9/9 9/9 rhvr ii/iii 8/10 2/10 10/10 6/8 1/8 0/8 0/8 0/10 1/10 3/10 6/10 10/10 7/10 8/8 8/8 8/8 8/8 rhvr i/ii/iii 7/10 0/10 10/10 6/10 5/10 0/10 0/10 0/10 2/10 5/10 7/10 10/10 8/10 10/10 10/10 10/10 10/10 beaudette 9/10 1/10 10/10 4/9 4/9 1/9 0/9 0/10 2/10 4/10 6/10 10/10 6/10 9/9 9/9 9/9 9/9 ck/ch/ldl/091022 2/5 c 0/5 5/5 3/5 2/5 0/5 0/5 0/10 5/6 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 pbs 10/10 3/10 10/10 8/9 3/7 0/7 0/7 0/10 0/10 0/10 0/10 0/10 3/10 4/9 5/7 7/7 7/7 a viral rna was detected in oral swab samples by real-time rt-pcr and samples with a ct value less than 32 were considered positive. b days after challenge. c five birds died 7 days after inoculation with ck/ch/ldl/091022. previous studies have shown that the s protein has major effects on pathogenicity in coronaviruses (leparc-goffart et al., 1997; phillips et al., 2001) , 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/ 091022, thereby indicating that only exchanging the hvr sequences did not have major impacts on pathogenicity (figs. 5 and 6, sup. fig. 2 ). according to hodgson et al., the apathogenic nature of the recombinant ibv beaur-m41(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., 2004) . it is possible that the replicase gene also contributes to the pathogenicity of ibv (armesto et al., 2009; hodgson et al., 2004) . 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., 2008; sperry et al., 2005) . it is considered that the hvrs of the ibv s1 subunit may induce abundant virus neutralizing antibodies koch et al., 1990; moore et al., 1997; niesters et al., 1987) . 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 2 ). 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., 2017) . 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 s1 subunit and s2 subunit (ignjatovic and sapats, 2005; kusters et al., 1989; lenstra et al., 1989) . in particular cases, a very low number of critical amino acid changes is sufficient to greatly alter the antigenicity (cavanagh et al., 1992) , although this view does not apply in all cases (chen et al., 2015) . 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 (lx4-type) emerged in china and spread to asia (mahmood et al., 2011) , russia (bochkov et al., 2006) , and europe (beato et al., 2005; worthington et al., 2008) . in this study, using ck/ ch/ldl/091022 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., 2009 ). 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 strain with the corresponding fragments from a qx-like nephropathogenic isolate ck/ch/ ldl/091022 from china. the results showed that the heterogenous hvrs could weaken the capacity for adsorption 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recombination recombinant newcastle disease virus expressing the infectious bronchitis virus s1 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-41-k18), the provincial supported science foundation of heilongjiang province for the national key technology r&d program (gx16b003) and national "twelfth five-year" plan for science & technology support (2015bad12b03). 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/10.1016/j.virusres.2018.04.013. key: cord-271638-0wsyl7vk 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/pi3k/akt pathway date: 2020-03-09 journal: antiviral res doi: 10.1016/j.antiviral.2020.104714 sha: doc_id: 271638 cord_uid: 0wsyl7vk 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-1 and hsv-2 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/pi3k/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/pi3k/akt pathway. herpes simplex virus type 1 (hsv-1) and type 2 (hsv-2) are enveloped double stranded dna viruses belonging to herpesviridae (fatahzadeh and schwartz, 2007) . hsv infection causes skin lesions that are generally localized at the oral, nasal, and ocular level with hsv-1 infection, whereas with hsv-2, infections most commonly occur at genital-skin and mucosa sites (liu and cohen, 2015; smith and robinson, 2002) . approximately 60%-90% of the adult human population worldwide is sera-positive for hsv-1 (carr et al., 2001) . 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, 2003) . hence, the development of novel anti-hsv agents with active mechanisms different from nucleoside analogues is of high importance. myricetin (3,5,7-trihydroxy-2-(3,4 ,5-trihydroxyphenyl)-4-benzopyrone) is a common dietary flavonoid from plant sources such as vegetables, fruits, and tea, with antioxidant properties (ong and khoo, 1997; ross and kasum, 2002) . 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, 2005; dajas et al., 2003; gupta et al., 2014; ong and khoo, 1997; santhakumar et al., 2014; semwal et al., 2016; tian et al., 2010) . pasetto et al. reported that myricetin possessed anti-hiv-1 activities with low toxicity, and it mainly inhibited the activity of hiv reverse transcriptase (pasetto et al., 2014) . lyu et al. found that some flavonoids such as myricetin and quercetinin showed moderate inhibitory effects against hsv-1 in vero cells (lyu et al., 2005) . 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., 2012) . 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-1 and hsv-2 activities with very low toxicity. myricetin may block hsv https://doi.org/10.1016/j.antiviral.2020.104714 received 5 october 2019; received in revised form 23 december 2019; accepted 20 january 2020 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 > 95%) was purchased from topscience co., ltd. (shanghai, china) . acyclovir was purchased from sigma aldrich (st. louis, mo, usa). vero, hela and hep-2 cells were routinely cultured in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum (excell bio, china), penicillin (100 u/ml), and streptomycin (100 μg/ml) at 37°c in 5% co 2 . hsv-1 strain f was purchased from atcc (vr-734). hsv-2 strain 333 was obtained from wuhan institute of virology, chinese academy of sciences. the antiviral activity was evaluated by the cpe inhibition assay (dai et al., 2018) . briefly, vero cells in 96-well plates were infected with hsv-1 or hsv-2 at a multiplicity of infection (moi) of 0.1, and then treated with indicated concentrations of myricetin in triplicate after removal of virus inoculum. after 24 h incubation, the cells were fixed with 4% formaldehyde for 20 min at room temperature (rt). after removal of the formaldehyde, the cells were stained with 0.1% (w/v) crystal violet for 30 min at 37°c. the plates were washed and dried, and the intensity of crystal violet staining for each well was measured at 570 nm. the concentration required for a test compound to reduce the cpe of hsv by 50% (ic 50 ) was determined. hsv-1 or hsv-2 (50-100 pfu/well) was pre-incubated with or without myricetin for 60 min at 37°c before infection. then the virusmyricetin mixture was transferred to vero cell monolayers in 6-well plates, and incubated at 37°c for 1 h with gentle shaking every 15 min. after that, the inoculum was removed and each well was overlaid with 2 ml of agar overlay media containing 1.5% agarose, 100 u/ml penicillin, and 100 μg/ml streptomycin. the cells were then incubated at 37°c until plaque sizes were adequate. then the cells were fixed with 4% paraformaldehyde (pfa) and stained with 1% crystal violet for plaque counting. vero cells were infected with hsv-1 or hsv-2 (moi = 1.0) under four different treatment conditions: i) pretreatment of virus: myricetin (20 μm) pretreated hsv was added to vero cells and incubated at 37°c for 1 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 20 μm of myricetin before hsv infection. iii) adsorption: vero cells were infected in media containing myricetin (20 μm) at 4°c for 1 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 (20 μm) was added to the cells. at 24 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-1 (moi = 1.0) was adsorbed to hela cells for 2 h at 4°c in the absence or presence of 20 μm myricetin before washing with pbs. for entry assay, hsv-1 (moi = 1.0) was firstly adsorbed to hela cells at 4°c for 2 h before washing with pbs. then myricetin (20 μm) was added to cells and incubated at 37°c for 1 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 4% paraformaldehyde for 20 min. then cells were permeabilized using 0.5% (v/ v) triton x-100 in pbs for 5 min before incubated with 2% bsa for 1 h at 37°c. after washing, cells were incubated consecutively with anti-icp5 antibodies (1:100 dilutions) and dylight 649®-conjugated secondary antibodies. then the cell nucleus was stained with dapi for 20 min before confocal imaging. images were recorded using a nikon confocal microscope, and analysed by imagej (nih) version 1.33 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, 5′-agcatcccgatcactgtgtacta-3′ and 5′-gcgatggtcaggttgt acgt-3'; monkey β-actin mrna, 5′-ctccatcctggcctcgctgt-3′ and 5′-gctgtcaccttcaccgttcc-3'. the real-time rt-pcr was performed at 42°c 5 min, 95°c 10 s, 40 cycles of 95°c 5 s, 60°c 34 s, followed by melting curve analysis, according to the instrument documentation (abi prism 7500, 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 (2 -δδct ) method, as previously described (livak and schmittgen, 2001) . darts experiments for identifying the targets of myricetin were performed as previously reported (lomenick et al., 2009 ). briefly, hsv-1 (moi = 1.0) infected vero cells were lysed with np-40 cell lysis buffer (beyotime, nantong, china) and treated with or without myricetin (60 μm) for 1 h at room temperature (rt) followed by digested with pronase (3 μg/ml) in reaction buffer (50 mm tris-hcl (ph 8.0), 50 mm nacl, 10 mm cacl2) for 30 min at rt. the digestion was stopped by directly add 5 × sds-page loading buffer and inactivation by boiling. protein samples were separated with 10% sds-polyacrylamide gels and analysed by western blot with antibodies against hsv-1 gd, gb, gc, gh/gl proteins or cellular hvem, nectin-1, actin, tubulin proteins as controls. spr assays were conducted on a spr biosensor instrument ge biacoret200 (ge, usa). hsv-1 gd proteins (biodragon, beijing, china) were firstly immobilized onto the surface of a carboxymethylated dextran sensor chip (cm5) via amino group coupling as described previously (geng et al., 2003) . to assess real-time binding of myricetin to the gd proteins on cm5 chips, myricetin with different concentrations (10, 5, 2.5, 1.25, 0.625, 0.3125 μm) dissolved in dmso, was injected over the sensor chip surface with gd immobilized within 2 min, followed by a 10-min wash with 1 × pbst buffer. the sensor chip surface was then regenerated by washing with naoh (2 mm) for 30 s. all binding experiments were carried out at 25°c with a constant flow rate of 2 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 biacoret200 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 177 (2020) 104714 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 0.1% tween 20 (v/v) and 5% bsa (w/ v) at rt for 2 h, the membranes were rinsed and incubated at 4°c overnight with primary antibodies against phosphorylated or nonphosphorylated egfr, pi3k, 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 (1:2000 dilutions) at rt for 2 h. the protein bands were then visualized by incubating with the developing solution (pnitro blue tetrazolium chloride (nbt) and 5-bromo-4-chloro-3-indolyl phosphate toluidine (bcip)) at rt for 30 min. the relative densities of proteins were all determined by using imagej (nih) v.1.33 u (usa). the cell fusion inhibition assay was performed as described with modifications (du et al., 2017) . monolayers of vero cells grown in 6well plates were infected with hsv-2 (moi = 3.0) at 4°c for 2 h. after removal of virus inoculum, compound free media were added to cells and incubated at 37°c for 5 h. then myricetin (30 or 15 μm) was added to cells, and incubated at 37°c for another 2 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 5-7 h post infection was also evaluated by western blotting. molecular docking was conducted in moe v2014.09011 (moe, 2014) . the 2d structures of myricetin was drawn in chembiodraw 2014 and converted to 3d structure in moe through energy minimization. the 3d structure of the protein gd was downloaded from rcsb protein data bank (pdb id: 4myv). prior to docking, the force field of amber10: 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 10. 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 20 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, 11.0 ± 2.0 g) were purchased from beijing vital river laboratory animal technology co., ltd. (beijing, china) and raised in a pathogen-free environment (23 ± 2°c and 55% ± 5% humidity). mice were randomly divided into five experimental groups (10 mice each). mice were anaesthetized and infected via the intranasal route with 10 6 pfu of hsv-1 (f strain) in 50 μl of pbs. four hours after inoculation, mice received intraperitoneally treatment of acyclovir (10 mg·kg-1), myricetin (2.5 or 5 mg·kg-1), or placebo, and the treatments were repeated once daily for five days. each day mice were weighed and monitored for signs of illness for 14 days, and those suffering a severe infection or having lost > 20% 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 4 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 4. 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 7 software using oneway anova with turkey's test. p values < 0.05 were considered significant. myricetin is a member of the flavonoid class of polyphenolic compounds, with antioxidant properties, and its structure is shown in fig. 1a . herein, the anti-hsv effects and mechanisms of myricetin were investigated in vitro. the cytotoxicity of myricetin in vero, hep-2 and hela cells was firstly assessed by mtt assay (livak and schmittgen, 2001) . the results showed that myricetin exhibited no significant cytotoxicity at the concentrations from 50 to 400 μm (fig. 1b) . the cc 50 (50% cytotoxicity concentration) value for myricetin in vero, hep-2 and hela cells was about 1097.9 ± 71.2 μm, 964.6 ± 94.7 μm, and 1152.3 ± 165.1 μm, respectively (table 1) . myricetin was then assayed for its ability to inhibit hsv multiplication in vitro using cpe inhibition assay and plaque assay . briefly, hsv-1 (f strain) or hsv-2 (333 strain) (moi = 0.1) was pretreated with myricetin (1.25, 2.5, 5, 10 μm) for 1 h at 37°c before infection, then after adsorption, the media containing myricetin at indicated concentrations were added to cells. at 24 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. 1c , myricetin significantly reduced the virus titers of both hsv-1 and hsv-2 in vero cells when used at the concentration > 1.25 μm (p < 0.05). cpe inhibition assay showed that the ic 50 values of myricetin was about 2.2 ± 0.3 μm and 1.6 ± 0.5 μm for hsv-1 and hsv-2, respectively, and the selectivity index (cc 50 /ic 50 ) for myricetin was about 498.6 and 685.6, respectively, superior to that of acyclovir (acv) (si = 131.8 and 84.1) ( table 1) . myricetin also inhibited hsv replication in hep-2 and hela cells with si > 150 (table 1 ). in addition, the plaque reduction assay showed that pretreatment of hsv with myricetin at the concentrations of 1.25-20 μm significantly reduced the number of plaques in both hsv-1 and hsv-2 infected cells (fig. 1d-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. 2a , pretreatment of hsv-1 or hsv-2 with 20 μm myricetin for 1 h before infection significantly reduced the virus titers of hsv, compared to the non-treated virus control group (p < 0.01), suggesting that myricetin may have direct w. li, et al. antiviral research 177 (2020) 104714 interaction with hsv particles. treatment of myricetin during adsorption or after adsorption also possessed obvious inhibition on virus multiplication (p < 0.05) ( fig. 2a) . however, pretreatment of cells did not significantly reduce virus titers in vitro ( fig. 2a) , suggesting that myricetin may not interact with vero cells directly. moreover, the indirect immunofluorescence assay under these four conditions (pachota et al., 2017) indicated that pretreatment of virus with myricetin (10 μm) before infection also significantly reduced the expression of icp27 in hsv-1 infected cells (fig. 2b) , which was in concern with the results in plaque assay ( fig. 2a) . 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-1 (moi = 1.0) was adsorbed to hela cells for 2 h at 4°c in the absence or presence of 20 μm myricetin before washing with pbs. for entry assay, hsv-1 (moi = 1.0) was firstly adsorbed to hela cells at 4°c for 2 h before washing with pbs. then myricetin (20 μm) was added to cells and incubated at 37°c for 1 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. 2c and e, myricetin treatment (20 μm) during adsorption significantly decreased the fluorescence of icp5 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 1 h p.i., many fluorescence spot of icp5 could be found at the nucleus area (white arrow indicated), suggesting that hsv-1 nucleocapsid had been egressed from late endosomes and delivered to the nuclear periphery in hela cells (fig. 2d) . however, myricetin treatment during entry process dramatically reduced the fluorescence of icp5 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. 2d, 2f and 2g ). 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. 3a , in hsv-2 (moi = 3.0) infected vero cells, obvious syncytia with multinuclear cells were observed in non-treated virus control group (hsv-2) at 7 h p.i. however, treatment with myricetin (30, 15 μm) during 5-7 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 5-7 h p.i. did not obviously influence the expression of virus surface glycoproteins such as gd protein (fig. 3b) . 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., 2009) , 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-1 infected vero cell lysates were digested with pronase in the presence or absence of myricetin (60 μm), then the amount of gd protein in cell lysates was detected by western blotting. as shown in fig. 3c , hsv-1 gd protein was obviously protected by protease digestion in the extracts of myricetin (60 μm) treated cells especially under pronase treatment at 3 μg/ml, suggesting that myricetin may interact with hsv gd protein in hsv-1 infected vero cells. however, myricetin (60 μm) could not protect other virus surface glycoproteins such as gb, gc, and gh/gl proteins from protease digestion in hsv-1 infected cells (fig. 3d) . similarly, the cellular surface receptors of hsv-1 such as hvem and nectin-1 could also not be protected from protease digestion by myricetin (60 μm) (fig. 3d) . 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., 2003) . briefly, with hsv-1 gd proteins being immobilized on the chip, myricetin at the concentrations of 0.3125-10 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 6.088e-8 m (60.88 nm), implicating a high affinity of myricetin for hsv-1 gd protein (fig. 3e) . thus, pretreatment of hsv were infected with hsv-1 or hsv-2 (moi = 1.0) using four different treatment conditions. i) pre +virus: hsv was pretreated with myricetin (20 μm) at 37°c for 1 h before infection. ii) pre+cell: vero cells were pretreated with 20 μm of myricetin before infection. iii) adsorption: vero cells were infected in media containing myricetin (20 μm) at 4°c for 1 h. iv) post-adsorption: after removal of unabsorbed virus, myricetin (20 μm) was added to the cells. at 24 h p.i., virus yields were determined by plaque assay. the results were presented as mean ± s.d. from five independent experiments. *p < 0.05 vs. virus control group. (b) vero cells were infected with hsv-1 (f strain) under four treatment conditions of myricetin, and the expression of virus icp27 protein was detected by immunofluorescence assay. scale bar represents 50 μm. (c and d) hsv-1 (moi = 1.0) infected hela cells were treated with or without myricetin (20 μm) during adsorption process (c) or entry process (d), then after washing three times with pbs, the immunofluorescence assay was performed by using anti-icp5 antibody to evaluate the amount of virions binding to or entering into cells. scale bar represents 50 μm. (e-g) the average fluorescence intensity of icp5 proteins during adsorption process (e) or entry process (f) was measured by imagej (nih) version 1.33u (usa) to calculate the average intensity per cell of different images (n = 10). the average intensity of icp5 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 = 10). the average intensity for nontreated virus control cells (hsv) was assigned values of 100 and the data presented as mean ± s.d. (n = 3). significance: **p < 0.01 vs. virus control group (hsv). w. li, et al. antiviral research 177 (2020) 104714 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-1 gd binding to vero cells was further explored using a cell enzyme-linked immunosorbent assay, as previously described (tiwari et al., 2006) . briefly, vero cells were first treated with or without myricetin (40, 20, 10, 5 μm) for 1 h, then the hsv-1 gd protein or myricetin (40, 20, 10, 5 μm) pretreated gd protein (2 μg/ml) were added to cells and incubated for another 1 h, the hsv-1 gd proteins were firstly immobilized onto the surface of a carboxymethylated dextran sensor chip (cm5). to assess realtime binding of myricetin to the gd proteins on cm5 chips, myricetin (10, 5, 2.5, 1.25, 0.625, 0.3125 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-1 gd binding assay. vero cells were first treated with or without myricetin (40, 20, 10, 5 μm) for 1 h, then the hsv-1 gd protein or myricetin (40, 20, 10, 5 μm) pretreated gd protein (2 μg/ml) were added to cells and incubated for another 1 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 = 3). significance: **p < 0.01 vs. virus control group. (g and h) the 2d (g) and 3d (h) binding mode of myricetin with hsv gd protein (pdb id: 4myv) 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 177 (2020) 104714 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. 3f , pre-incubation of gd protein with myricetin (5, 10, 20, 40 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-1 gd protein (pdb code: 4myv). 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 gln41 as well as asn94 in gd (fig. 3g) . 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 leu44 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 glu45 in gd ( fig. 3g and h) . thus, myricetin may interact with gln41, leu44, glu45 and asn94 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/ pi3k/akt signaling pathway was reported to be required for virus endocytosis and replication, and the inhibitors of pi3k signaling could inhibit both entry and fusion of hsv (eierhoff et al., 2010; tiwari and shukla, 2010) . herein, treatment with myricetin (10, 20 μm) for 2 h significantly decreased the levels of phosphorylated egfr from about 2.4 to about 1.4 and 0.8-fold of normal control group, respectively (p < 0.05) (fig. 4a and d) . moreover, the activation of downstream signal pi3k was also evaluated, and the results showed that treatment with myricetin (5, 10, 20 μm) for 2 h significantly decreased the levels of phosphorylated pi3k proteins from about 1.9 to about 1.4, 1.4, and 0.8-fold of normal control group, respectively (p < 0.01) (fig. 4b and e). in addition, treatment with myricetin (5, 10, 20 μm) for 2 h significantly reduced the activation of akt from about 12.1 to about 3.9, 2.5, and 1.2-fold of normal control group, respectively (p < 0.05) ( fig. 4c and f) . however, myricetin (2.5, 5, 10, 20 μm) treatment did not significantly influence the total expression levels of egfr, pi3k and akt proteins in hsv-2 infected vero cells (fig. 4g and h) . thus, cellular egfr/pi3k/akt signaling pathway may be involved in the anti-hsv fig. 4 . involvement of egfr/pi3k/akt signaling pathway in the anti-hsv actions of myricetin. (a-c) hsv-2 (moi = 1.0) infected cells were treated with or without myricetin (2.5, 5, 10, 20 μm) for 2 h, and then the phosphorylation of egfr (a), pi3k (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-pi3k (e), p-akt (f) protein to gapdh or tubulin, respectively. the ratio for non-infected cells (m) was assigned values of 1.0 and the data presented as mean ± s.d. (n = 3). significance: ##p < 0.01 vs. normal control group (mock); *p < 0.05, **p < 0.01 vs. virus control group (hsv). (g) hsv-2 (moi = 1.0) infected cells were treated with or without myricetin (2.5, 5, 10, 20 μm) for 2 h, and then the total expression levels of egfr, pi3k, 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, pi3k, akt protein to α-tubulin, respectively. the ratio for non-infected cells (mock) was assigned values of 1.0 and the data presented as mean ± s.d. (n = 3). w. li, et al. antiviral research 177 (2020) 104714 actions of myricetin in vitro. furthermore, myricetin (2.5, 5, 10, 20 μm) treatment could not significantly influence the activation of egfr, pi3k and akt proteins in the non-infected vero cells ( fig. 5a and b) , suggesting that the inhibition of egfr/pi3k/akt pathway by myricetin may be related to its inhibition of hsv infection. moreover, treatment with hsv-1 gd protein (2 μg/ml) could significantly enhance the phosphorylation of egfr, pi3k, and akt proteins in the non-infected vero cells (p < 0.01), suggesting that the activation of egfr/pi3k/akt pathway in hsv infected cells may be related to the binding of gd to cellular receptors ( fig. 5c and d) . however, after myricetin treatment (5, 10, 20 μm), the levels of phosphorylated egfr, pi3k, and akt proteins were significantly reduced as compared to the gd treated control group (p < 0.05) ( fig. 5c and d) . in addition, myricetin treatment (5, 10, 20 μm) also significantly reduced the expression level of gd mrna in hsv-1 and hsv-2 infected vero cells (p < 0.05) ( fig. 5e and f) . considered that egfr/pi3k/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/pi3k/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, 1976) . in brief, hsv-1 (f strain)-infected mice received intraperitoneal treatment of myricetin (2.5 or 5 mg·kg-1), acyclovir (10 mg·kg-1), or placebo (pbs) once daily for five days. as shown in fig. 6a , intraperitoneal treatment of myricetin (2.5 or 5 mg·kg-1) significantly increased survival rates as compared to the placebotreated control group (p < 0.01). by day 14 post infection, only 20% of the individuals in the placebo group survived whereas 100% of animals in the myricetin-treated group survived, superior to that in acyclovir (10 mg·kg-1)-treated group (90%). moreover, the body weights of mice in virus control group (placebo) began to decrease at 6 days p.i., losing up to 19% of initial weight, before gradually recovering. however, myricetin (2.5 or 5 mg·kg-1)-treated mice gradually increased their body weights only with a limited weight loss of less than 5% during the infection, superior to those in the acyclovir (10 mg·kg-1) treated group (fig. 6b) . 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 < 0.05) (fig. 6c ). 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. 6d) . acyclovir (10 mg·kg-1) treatment also showed significant reduction of virus titers in mice lungs and spinal cord ( fig. 6c 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. 6e) . however, mice treated with myricetin (2.5 or 5 mg·kg-1) following infection had intact columnar epithelium even in the presence of tiny serocellular exudates in the lumen fig. 5 . the inhibition of egfr/pi3k/akt pathway by myricetin may be related to its inhibition of gd protein. (a) non-infected vero cells were treated with or without myricetin (2.5, 5, 10, 20 μm) for 2 h, and then the phosphorylation of egfr, pi3k, 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-pi3k and p-akt proteins. the ratios for non-treated cells (mock) were assigned values of 1.0 and the data presented as mean ± s.d. (n = 3). (c) vero cells were treated with or without gd proteins in the presene or absence of myricetin (5, 10, 20 μm) for 2 h, and then the phosphorylation of egfr, pi3k, 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-pi3k, p-akt protein to gapdh, respectively. the ratio for non-treated control cells (mock) was assigned values of 1.0 and the data presented as mean ± s.d. (n = 3). significance: ##p < 0.01 vs. normal control group (mock); *p < 0.05, **p < 0.01 vs. gd treated control group (gd). (e and f) hsv (moi = 1.0) infected vero cells were treated with myricetin (2.5, 5, 10, 20 μm), and incubated at 37°c for 8 h. after that, total rna was extracted for real-time rt-pcr assay of hsv-1 (e) and hsv-2 (f) gd mrnas and cellular β-actin mrna. the relative amounts of hsv gd mrnas were determined using the comparative (2 -δδct ) method. rna levels for non-treated virus control cells (hsv) were assigned values of 1.0. values are means ± sd (n = 3). significance: *p < 0.05, **p < 0.01 vs. virus control group. w. li, et al. antiviral research 177 (2020) 104714 fig. 6. the anti-hsv actions of myricetin in vivo. (a) survival rate. hsv-1 infected mice were received intraperitoneally treatment of myricetin (2.5 or 5 mg·kg-1), acyclovir (10 mg·kg-1), or placebo (pbs) once daily for five days. results are expressed as percentage of survival, evaluated daily for 14 days. significance: *p < 0.05, **p < 0.01 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 14 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 4 p.i., respectively. values are means ± s.d. (n = 4). significance: *p < 0.05, **p < 0.01 vs. virus control group. (e and f) histopathology analyses of lung and brain tissues on day 4 p.i. by he staining ( × 10 and × 20). the representative micrographs from each group were shown. mock: non-infected mice tissues; hsv-1: hsv-1 infected tissues without drugs; hsv-1+acyclovir-10: hsv-1 infected tissues with acyclovir (10 mg·kg-1) treatment; hsv-1+myricetin-5: hsv-1 infected tissues with myricetin (5 mg·kg-1) treatment; hsv-1+myricetin-2.5: hsv-1 infected tissues with myricetin (2.5 mg·kg-1) treatment; the black arrows indicate the presence of hyperaemia and infiltration of inflammatory cells in the lumen. ( fig. 6e ). in addition, treatment of myricetin (5 mg·kg-1) nearly had no hyperaemia and infiltration of inflammatory cells in brain tissues (fig. 6f) . myricetin (2.5 mg·kg-1) treated mice only had tiny hyperaemia in the brain tissues, comparable to the effects of acyclovir (10 mg·kg-1) (fig. 6f) . 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, 1997) . in this study, we discovered that myricetin possessed anti-hsv-1 and hsv-2 activities in different cells with very low toxicity (si > 150). 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-1-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. 3) . the data from spr assay verified that myricetin can truly bind to hsv-1 gd protein with high affinity (kd ≈ 60.88 nm) (fig. 3e) . the molecular docking analysis indicated that myricetin may interact with gln41, leu44, glu45 and asn94 of gd protein through hydrogen bond interactions ( fig. 3g and h) . furthermore, myricetin treatment inhibited the activation of egfr, pi3k 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. 5) . considered that egfr/pi3k/akt pathway is required for efficient hsv replication (tiwari and shukla, 2010) , we suppose that myricetin may interact with virus gd protein to interfere with the activation of egfr/pi3k/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., 2004) . in this study, intraperitoneal therapy of hsv-1-infected mice with myricetin markedly improved their survival and inhibited hsv multiplication in both lungs and spinal cord (fig. 6) . 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/ pi3k/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/pi3k/akt pathway. further studies of the anti-hsv effects of myricetin 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inhibitors of the sars coronavirus helicase, nsp13 this work was supported by national natural science foundation of china (81874320, 81741146, 31500646), nsfc-shandong joint fund (u1606403, u1706210), shandong provincial natural science foundation (zr2017mh013), and qingdao marine biomedical science and technology innovation center (2017-cxzx01-3-11). supplementary data to this article can be found online at https:// doi.org/10.1016/j.antiviral.2020.104714. key: cord-260107-gqbtkf0x authors: lee, sunhee; kim, youngnam; lee, changhee title: isolation and characterization of a korean porcine epidemic diarrhea virus strain knu-141112 date: 2015-10-02 journal: virus res doi: 10.1016/j.virusres.2015.07.010 sha: doc_id: 260107 cord_uid: gqbtkf0x 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-141112/2014, was successfully isolated and serially propagated in vero cells for over 30 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 30 passages ranged from 10(5.1) to 10(8.2) tcid(50) per ml. the inactivated knu-141112 virus was found to mediate potent neutralizing antibody responses in immunized guinea pigs. animal studies showed that knu-141112 virus causes severe diarrhea and vomiting, fecal shedding, and acute atrophic enteritis, indicating that strain knu-141112 is highly enteropathogenic in the natural host. in addition, the entire genomes or complete s genes of knu-141112 viruses at selected cell culture passages were sequenced to assess the genetic stability and relatedness. our genomic analyses indicated that the korean isolate knu-141112 is genetically stable during the first 30 passages in cell culture and is grouped within subgroup g2b 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, 1980; saif et al., 2012; pijpers et al., 1993) . the disease was initially recognized in england in 1971 and has then spread to swine-producing european countries (oldham, 1972; pensaert et al., 1981) . since the 1990s, ped has become rare in europe and is more often associated with post-weaning diarrhea in adult pigs (saif et al., 2012) . ped was first reported in asia in 1982 and has since had a great economic impact on the asian pork industry (chen et al., 2008; kweon et al., 1993; li et al., 2012; puranaveja et al., 2009; takahashi et al., 1983) . in may 2013, 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, 2013; stevenson et al., 2013 vlasova et al., 2014 . the etiological agent of ped, ped virus (pedv), was identified as a coronavirus in 1978, which is a member of the genus alphacoronavirus within the family coronaviridae of the order nidovirales (lai et al., 2007; pensaert and de bouck, 1978; saif et al., 2012) . pedv is a large, enveloped virus possessing a single-stranded positivesense rna genome of approximately 28 kb with a 5 cap and a 3 polyadenylated tail (pensaert and de bouck, 1978; saif et al., 2012) . 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., 2001; lai et al., 2007; lee et al., 2010) . 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., 2014; gerber et al., 2014; lee et al., 2010; lee and lee, 2014; oh et al., 2014) . the first ped epizootic in korea was confirmed in 1992 (kweon et al., 1993) . however, a retrospective study revealed that pedv already existed as early as 1987 (park and lee, 1997) . since the emergence, ped outbreaks occurred every year, resulting in substantial economic losses to the korean swine industry until early 2010. after severe outbreaks of foot-and-mouth disease (fmd) during 2010 to 2011, 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 2010-2011 fmd outbreaks. however, starting in november 2013, severe ped epidemics re-emerged in korea and swept more than 40% of pig farms lee et al., 2014a,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., 2010; oh et al., 2014; lee and lee, 2014) . 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., 2014) . 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., 1999; song et al., 2003) , 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., 2014; oka et al., 2014) , 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-141112/2014 has been successfully isolated and serially propagated in cell culture for over 30 passages. we aimed to characterize the growth and titer of the pedv isolate knu-141112 during the serial passages and the pathogenicity of the virus in suckling piglets. our in vivo assessment demonstrated that knu-141112 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-141112 were determined at selected passages to study the genetic stability and relationship. our data indicated that knu-141112 isolate is relatively stable during the first 30 passages in cell culture and is classified into subgroup g2b that includes pedv strains responsible for recent severe outbreaks in korea and the us. vero cells (atcc ccl-81) were cultured in alpha minimum essential medium (␣-mem; invitrogen, carlsbad, ca) with 5% fetal bovine serum (fbs; invitrogen) and antibiotic-antimycotic solutions (100×; invitrogen) and maintained at 37 • c in a humidified 5% co 2 incubator. seven small intestinal homogenates and 50 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 10% (wt/vol) suspensions with phosphate-buffered saline (pbs) using a magna lyser (roche diagnostics, mannheim, germany) by three repetitions of 15 s at a speed of 7000 rpm. fecal samples were diluted with pbs to be 10% (wt/vol) suspensions. the suspensions were then vortexed and centrifuged for 10 min at 4500 × g (hanil centrifuge fleta5, incheon, south korea). the supernatant was filtered through a 0.22-m-pore-size syringe filter (millipore, billerica, ma) and stored at −80 • 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, 1988) . 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 10 g/ml. confluent vero cells grown in 6-well plates were washed with pbs and inoculated with 400 l of each inoculum containing trypsin. after incubating at 37 • c for 1 h, 2 ml of virus growth medium [␣-mem supplemented with antibioticantimycotic solutions, 0.3% tryptose phosphate broth (tpb; sigma, st. louis, mo), 0.02% yeast extract (difco, detroit, mi), 10 mm hepes (invitrogen), and 5 g/ml of trypsin] was added. the inoculated cells were maintained at 37 • c under 5% co 2 and monitored daily for cytopathic effects (cpe). when 70% cpe appeared, inoculated cells were subjected to three rounds of freezing and thawing. the culture supernatants were then collected and centrifuged at 400 × g for 10 min. the clarified supernatants were aliquoted and stored at −80 • c as 'passage 1 (p1)' 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 7 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 6 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-141112 virus stock in the presence of trypsin as described above. the culture supernatants were collected at 24 or 48 h postinfection (hpi) at which a 70% cpe is commonly developed. for growth kinetics experiments, the supernatants were harvested from cells infected with each selected passage virus at different time points (6, 12, 24, 36, and 48 hpi) and stored at −80 • c. virus titers were measured in 96-well plates by 10-fold serial dilution of the samples in triplicate per dilution to determine the quantity of viruses required to produce cpe in 50% of inoculated vero cells and calculated as tcid 50 per ml using the reed-muench method (reed and muench, 1938) . 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 6-well tissue culture plates were mock infected or infected with pedv at a multiplicity of infection (moi) of 0.1. the virus-infected cells were subsequently propagated until 24 hpi, fixed with 4% paraformaldehyde for 10 min at room temperature (rt) and permeabilized with 0.2% triton x-100 in pbs at rt for 10 min. the cells were blocked with 1% bovine serum albumin (bsa) in pbs for 30 min at rt and then incubated with n-specific mab for 2 h. after being washed five times in pbs, the cells were incubated for 1 h at rt with a goat anti-mouse secondary antibody conjugated to alexa fluor 488 (invitrogen), followed by counterstaining with 4 ,6-diamidino-2-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., 2007; sagong and lee, 2011) . 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, 2011) . eight 3-4 month-old guinea pigs (weighing 300-350 g) were randomly allocated into inoculated (n = 6) and control (n = 2) groups. six guinea pigs were immunized subcutaneously with 0.5 ml of binary ethylenimine (bei)-inactivated knu-141112-p10 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 2-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 2 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 96-well microtiter plates using pedv isolate knu-141112 or vaccine strain sm98-1 as previously described (oh et al., 2014) with minor modifications. briefly, vero cells were grown at 2 × 10 4 /well in 96-well tissue culture plates for 1 day. the knu-141112-p10 virus stock was diluted in serum-free ␣-mem to make 200 tcid in a 50 l volume. the diluted virus was then mixed with 50 l of 2-fold serial dilutions of individual inactivated sera in 96-well plates and incubated at 37 • c for 1 h. the mixture was inoculated into vero cells and incubated at 37 • c for 1 h. after removing the mixture, the cells were thoroughly rinsed 5 times with pbs and maintained in virus growth medium at 37 • c in a 5% co 2 incubator for 2 days. for the sn test using the vaccine strain, pedv strain sm98-1 propagated in the absence of trypsin was diluted in serum-free ␣-mem to make 200 pfu in a 50 l volume. the diluted virus was then mixed with each serum sample in 96-well plates as described above and incubated at 37 • c for 1 h. subsequently, approximately 1 × 10 4 vero cells in 100 l of ␣-mem containing 5% fbs were added to each well and the mixture was maintained at 37 • c in a 5% co 2 incubator for 3 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 8 suckling piglets of 7 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 3 experimental groups housed in 2 separated rooms: pedv-inoculated (n = 4) and contact control (n = 2) in room 1 and sham-inoculated control (n = 2) group in room 2. following a 1 day acclimation period, only piglets in the pedv-inoculated group orally received a 1 ml dose of 10 3 tcid 50 /ml of knu-141112-p10 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 16 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 1, 2, 3, and 4) after challenge for post-mortem examinations throughout the study, whereas all pigs from the contact and control groups were euthanized at 4 days post-challenge for post-mortem examinations. small intestinal tissue specimens (<3 mm thick) collected from each piglet were fixed with 10% formalin for 24 h at rt and embedded in paraffin according to standard laboratory procedures. the formalin-fixed paraffin-embedded tissues were cut at 5-8 m thick on a microtome (leica), floated on a 40 • c water bath containing distilled water, and transferred onto glass slides. the tissues were then deparaffinized in xylene for 5 min and washed in decreasing concentrations of ethanol (100%, 95%, 90%, 80%, and 70%) for 3 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 0.01 m citrate buffer (ph 6.0) in a microwave oven for 5 min, chilled at rt for 20 min, and then incubated with 0.3% hydrogen peroxide in dw for 20 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 1 h at rt with n-specific mab. after rinsing in pbs, the samples were reacted for 45 min at rt with a horse anti-mouse secondary antibody (vectastain abc kit), incubated with avidin-biotin peroxidase complex for 45 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 p5 and p10 isolates were determined by nextgeneration sequencing (ngs) technology. total rna was extracted from the feces as well as p5 and p10 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., 2014b) . 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 316 v2 sequencing chip (life technologies) as described previously lee et al., 2014b; rothberg et al., 2011) . the single-nucleotide variants (snvs) were analyzed using the clc genomic workbench version 7.0 (clc bio, cambridge, ma) and the individual ngs reads were assembled using the complete genome of pedv reference strain kor/knu-1305/2013 (genbank accession no. kj662670). the 5 and 3 ends of the genomes of the original feces and the p5 and p10 isolates were determined by rapid amplification of cdna ends (race) as described previously . the full-length genomic nucleotide sequences of the knu-141112-feces, knu-141112-p5, and knu-141112-p10 were deposited in genbank under accession numbers kr873431, kr873434, and kr873435, 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., 2010) . 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 t7 and sp6 primers and the s gene-specific primers. in addition, the complete structural gene sequences of the virus isolate at selected passages (knu-141112-p3, knu-141112-p4, knu-141112-p20, and knu-141112-p30) were determined by the sanger method as described above and deposited to the gen-bank database under their accession numbers shown in fig. 5 . the sequences of 42 fully sequenced s genes and 25 complete genomes of pedv isolates were independently used in sequence alignments and phylogenetic analyses. the multiplesequencing alignments were generated with the clustalx 2.0 program (thompson et al., 1997) 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 1000 replicates in order to determine percentage reliability values on each internal node of the tree (saitou and nei, 1987) . all tree figures were produced using mega 4.0 software (tamura et al., 2007) . student's t test was used for all statistical analyses, and p-values of less than 0.05 were considered statistically significant. we attempted to isolate pedv from pcr-positive clinical samples, including 50 feces and 7 intestinal homogenates on vero cells. one pedv isolate designated knu-141112 was successfully isolated from the feces of a naturally infected piglet from a commercial farm located in kyungpook province obtained on september 29, 2014. the knu-141112 virus was capable of producing distinct cpes typical for pedv infection, such as cell fusion, syncytium, and detachment, in infected vero cells from passage 3 (p3). we then investigated whether the isolate can be efficiently cultivated and maintained in cell culture. thus, the isolated pedv strain knu-141112 was further serially passaged in vero cells for a total of 30 passages (p1 to p30). the time of cpe onset was at 24 hpi and, accordingly, prominent cpe was observed within 48 hpi in the first 2 productive passages (p3 and p4). in the later passages, visible cpe appeared at 12 hpi and became predominant by 24 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. 1 . 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 16.7, ranging from 15.3 (p10) to 18.7 (p5). the infectious titer of the isolate ranged from 10 5.1 to 10 6.1 tcid 50 /ml up to p5, whereas it was determined to be approximately 10 7 tcid 50 /ml in the later passages. the peak viral titer reached 10 7.8 tcid 50 /ml (equivalent to 10 7.5 pfu/ml) or more since passage 10 ( fig. 2a) . the growth kinetics study further showed that knu-141112 replicated rapidly and efficiently in vero cells, reaching a maximum titer >10 7 tcid 50 /ml by 24 dpi (fig. 2b) . guinea pig antisera were collected before immunization (preimmune) and at 2 weeks after the final boost and tested for their neutralizing activity against the isolate knu-141112 or vaccine strain. as shown in fig. 3 , the guinea pig antisera were highly effective in inhibiting knu-141112 infection with mean neutralizing antibody (na) titers of 1:112, whereas the antisera at relatively low dilution fully protect vero cells from sm98-1 infection with mean na titers of 1:37. 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-141112 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-141112 virus, while 2 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 1 day post-inoculation (dpi) and experienced severe watery diarrhea with vomiting thereafter. pedv-associated mortality occurred in one of the inoculated pigs at 1 dpi. contact piglets housed with the inoculated group exhibited diarrheic feces with vomiting by 2 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 1 or 2 dpi and shed pedv in feces with mean ct values of 18.7 (range 16.4-21.0) and 20.1 (range 15.4-23.1), 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 1 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. 4, 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. 4, panels b and c) . similar microscopic lesions were continuously present in the challenged and contact pigs through 4 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. 4 , 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 24 hpi using an inverted microscope at a magnification of 200× (first panels). for immunostaining, infected cells were fixed at 24 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 200× magnification. (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 2013). 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-141112-feces) and cell culture-passaged knu-141112-p5 and -p10 viruses were determined using the ngs technology. the full-length genome of kor/knu-1305/2013 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 5 and 3 ends of their genomes were also sequenced by race. all three genomes were 28,038 nucleotides (nt) in length, excluding the 3 poly(a) tail and exhibited the genomic organization typical of all previously sequenced pedv strains, consisting of the 292-nt 5 utr, the 20,345-nt orf1a/1b (nt 293 to 12,601 for 1a and nt 12,601 to 20,637 for 1b), the 4161-nt s gene (nt 20,634 to 24,794), the 675-nt orf3 (nt 24,794 to 25,468), the 231-nt e gene (nt 25,449 to 25,679) , the 681-nt membrane (m) gene (nt 25,687 to 26,367), the 1326-nt n gene (nt 26,379 to 27,704), and the 334fig. 3 . virus-neutralizing antibody titers in the sera of guinea pigs inoculated with a pedv knu-141112 isolate. guinea pigs were immunized twice with inactivated knu-141112-p10 virus by subcutaneous injection. blood samples were collected prior to immunization and at 2 weeks after the second immunization and subjected to the virus neutralization assay using homologous (knu-141112; solid circles) and heterologous (sm98-1; solid diamonds) strains. neutralizing antibody titers for individual infected animals were spotted as a log2 scale. values are representative of the mean from three independent experiments in duplicate and error bars denote standard deviations. nt 3 utr. the slippery heptamer sequence, tttaaac, followed by a stem-loop structure was present at the 3 end of orf1a in the pedv genome, which is a potential signal for a ribosomal frame shift during translation to generate the pp1ab. the complete genome sequences of all three viruses were compared, and the results are summarized in table 1 . compared to the knu-141112-feces, knu-141112-p5 showed one different nt at position 21,756 resulting in one amino acid (aa) change (leu to phe) in the s protein, while knu-141112-p10 gained two additional nt changes at positions 21,448 and 24,492 causing two aa mutations located in the s protein. the full-length s genes of the original feces and knu-141112 viruses at selected passages (p3, p4, p5, p10, p20, and p30) were also sequenced using the traditional sequencing method. the s protein sequences of knu-141112-feces, -p5, and -p10 were completely identical to those determined by ngs. one nt change present in the s gene of knu-141112-p5 had been acquired since passage 3 (knu-141112-p3). a total of three nt mutations, identified at passage 10 compared to the feces, were sustained through passage 30 (knu-141112-30) . in addition, compared to knu-141112-p10, we were able to detect two independent mutations at positions 24,869 and 25,656, resulting in amino acid changes in orf3 (asp to tyr) and e (pro to ser) of knu-141112-p20, respectively, which were maintained at passage 30. altogether, our results revealed that the pedv isolate knu-141112 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 3 other korean and 8 non-korean pedv strains available in genbank, and the nucleotide homology and difference data are described in table 2 . knu-141112 isolates (feces, p5, and p10) had the highest nucleotide identity (99.9%) with the korean re-emergent strain kor-knu-1305 and us strains, in17846 and mn, showing 8 to 46 different nt at the genomic level. all three viruses were genetically distinct from the korean vaccine strains, sm98-1 and dr-13, and the prototype cv777 strain and exhibited relatively low nucleotide identity ranging from 96.3% to 96.8%. by alignment of the global pedv strains, a single-nucleotide insertion between nucleotides 20,204 and 20,205 has been previously identified in one chinese ah2012 and three us strains, table 1 nucleotide and amino acid changes of knu-141112 during serial passages in cell culture. nucleotide amino acid position feces p3 p4 p5 p10 p20 p30 position feces p3 p4 p5 p10 p20 p30 5 utr (292) -a -n d b nd --nd nd --nd nd --nd nd orf1a (12,354) --nd nd --nd nd --nd nd --nd nd orf1ab (20,345) --n d nd --nd nd --nd nd --nd nd s (4,161) 21448 a a a a c c c 272 k k k k t t t 21756 c t t t t t t resulting in the shorter pp1ab protein in length (chen et al., 2014) . 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., 2010; lee and lee, 2014; lee et al., 2014a,b) , the full-length s gene-based phylogenetic tree revealed that the global pedv strains were clearly defined into 2 separate clusters, designated genogroup 1 (g1; classical) and genogroup 2 (g2; field epidemic). each genogroup can be further divided into subgroups 1a, 1b, 2a, and 2b (fig. 5a ). all original feces and passaged pedv viruses through passages 30 were classified into subgroup 2b 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 s1 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-141112 is grouped within the same cluster with the recent korean and us stains (fig. 5b ). in korea, three pedv strains, sm98-1, dr-13, and chinju99, were initially isolated almost two decades ago in korea. genetic and phylogenetic analyses revealed that sm98-1 and dr-13 belong to the classical group 1, whereas chinju99 is classified into the field epidemic group 2. of these, only sm98-1 and dr-13 isolates can be grown in cell culture and, accordingly, have been used as commercial vaccine seeds. since 2008, 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 10% amino acid difference in the s1 domain (lee et al., 2010; lee and lee, 2014; oh et al., 2014) . 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., 2014; oka et al., 2014) . in this study, we initially sought to isolate pedv efficiently propagated in vitro from 7 intestine homogenates and 50 fecal samples of naturally infected piglets (field cases) and were able to obtain only 1 isolate from feces in vero cell culture. the virus isolation rate, in the present study, was less than 2% and was relatively lower than that in recent us studies ranging from 5 to 10% (chen et al., 2014; oka et al., 2014) . 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., 2014; oka et al., 2014) . however, we failed to isolate pedv from intestine homogenates, which may be due to the number of intestinal contents (n = 7) 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-141112, was cytopathogenic in vero cells from passage 3 and after passage 5, exhibited more severe and rapid cpe characterized by fusion of infected cells (syncytium or polykaryon formation). the initial viral infectious titers ranged from approximately 5 to 6 log 10 tcid 50 /ml and increased after several more passages reaching to 8 log 10 tcid 50 /ml or more. these growth characteristics, including cytopathology, infectious titer, and growth kinetics were unchanged or even more efficient throughout the experiment (30 passages), indicating that the isolate knu-141112 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-141112-p10) 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 (6.4 log 2 ), demonstrating the ability of knu-141112 to elicit immune responses. on the other hand, the antisera showed a relatively weak neutralizing response (an almost 2-log 2 reduction), when the heterologous vaccine strain sm98-1 was used for an sn test. this weak neutralizing activity of the anti-knu-141112 guinea pig sera against sm98-1 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., 2010; lee and lee, 2014; oh et al., 2014) . experimental oral inoculation of nursery pigs with pedv isolate knu-141112 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 1 dpi. the pedv genome was detected in feces in 100% of challenged pigs on dpi 1 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 1 in only one-fourth of the challenged pigs (madson fig. 5 . 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 50% of 1000 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., 2014) . the difference between the current and previous studies is the age of the pigs: 1-week-old neonatal pigs and 3-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., 2000) . ihc and ifa revealed that viral antigen in villous enterocytes were observed at 1 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., 2014) . 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 24 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 100% morbidity in our study. mortality averages 50% in suckling piglets up to 1 week of age, often approaching 100% in 1-to 3-dayold piglets, and decreases to 10% thereafter (saif et al., 2012) . in our study, mortality was observed only in one out of four inoculated piglets, resulting in 25% mortality in the current study involving 8-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 3 korean pedv strains (knu-141112-feces, p5, and p10) 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-141112-feces), only one nt difference at position 21,756 was identified for the first 5 passages (knu-141112-p5) in cell culture, which led to a non-synonymous mutation at the corresponding position 375 of the s protein. this nt change was initially found at passage 3 and further maintained through passage 30. 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 c21756t mutation at the whole-genome level (l375f at the aa level of s) has been previously reported in a us pedv isolate isu-19338e during cell culture passage (chen et al., 2014) , suggesting its potential importance for adaption of the field virus to growth in vitro. at passage 10, two more nt differences at positions 21,448 and 24,492 of the genome (a21448c and g24492c) were detected when compared to knu-141112-feces. all of the changes acquired in knu-141112-p10 led to non-synonymous mutations at the respective positions 272 and 1287 of the s protein (k272t and e1287q). these findings were similar to recent data reported by chen et al. (2014) that two us isolates individually gained the 4 mutations located in orf1b, s, and e through passage 9 in cell culture. however, no nucleotide changes were identified in orf1b and e for the first 10 passages in vero cells. the 3 mutations in the s protein were persistent for 30 passages in cell culture. celladapted pedv vaccine strains, sm98-1 and dr-13, are known to contain a 52-nt deletion spanned from the end of s to the start of orf3 and a 49-nt deletion in the middle of orf3. thus, we further sequenced structural genes of knu-141112-p20 and -p30 and identified two additional nucleotide changes (g24869t and c25656t) acquiring non-synonymous amino acid mutations in orf3 and e, respectively. except for those nucleotide substitutions, the isolate had no extra change, including such large deletions in structural genes through passage 30, indicating the genetic stability of knu-141112 during serial passages in vero cells. sequence comparisons with other pedv strains showed that knu-141112 isolates are genetically most similar to recent korean and us epidemic strains with 99.8-99.9% identities, but most distinctly related to classical strains, cv777, sm98-1, and dr-13, with 96.3-96.8% identities at the genomic level. all phylogenetic analyses based on the complete genome, the full-length s gene, and the s1 portion formed the similar tree structure, revealing that the korean knu-141112 strains are clustered together with the re-emergent korean strains in subgroup 2b that also includes 2013 emergent us strains and 2011-2012 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 2013 disease outbreak among swine in the united states experimental infection of pigs with a new porcine enteric 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korea outbreak-related porcine epidemic diarrhea virus strains similar to us strains, south korea reemergence of porcine epidemic diarrhea virus on jeju island full-genome sequence analysis of a variant strain of porcine epidemic diarrhea virus in south korea complete genome sequence of a novel porcine parainfluenza virus 5 isolate in korea new variants of porcine epidemic diarrhea virus pathogenesis of porcine epidemic diarrhea virus isolate (us/iowa/18984/2013) in 3-week-old weaned pigs immunogenicity and protective efficacy of recombinant s1 domain of the porcine epidemic diarrhea virus spike protein cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene letter to the editor retrospective study of porcine epidemic diarrhea virus (pedv) in korea by in situ hybridization a new coronavirus-like particle associated with diarrhea in swine an immunoelectron microscopic and immunofluorescent study on the antigenic relationship between the coronavirus-like agent, cv777, 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 irf3 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 3 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 mega4: molecular evolutionary genetics analysis (mega) software version 4.0 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-2012r1a1a2039746). key: cord-254317-n2knqj4z authors: su, yunfang; hou, yixuan; wang, qiuhong title: the enhanced replication of an s-intact pedv during coinfection with an s1 ntd-del pedv in piglets date: 2018-11-27 journal: vet microbiol doi: 10.1016/j.vetmic.2018.11.025 sha: doc_id: 254317 cord_uid: n2knqj4z porcine epidemic diarrhea virus (pedv) variants having a large deletion in the n-terminal domain of the s1 subunit of spike (s) protein were designated as s1 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: icpc22a and its s1 ntd-del form icpc22a-s1δ197. 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 icpc22a and icpc22a-s1δ197, icpc22a replicated to a higher peak titer than its infection of pigs without the presence of icpc22a-s1δ197. the severity of diarrhea and intestinal atrophy were similar between icpc22a and the coinfection groups, but were significantly higher than icpc22a-s1δ197 group. in vero and ipec-dq cells, certain concentrations of bm, pgm, bile and bile acids increased significantly the infectivity of icpc22a but had no or negative effects on icpc22a-s1δ197. 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 s1 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 28 kb. it contains orf1a and orf1b encoding 14-16 non-structural proteins, an orf3 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 s1 subunit) and subsequent fusion of the viral and cellular membranes (via s2 subunit) (li, 2016) . s1 subunit contains two domains, the n-terminal domain (s1 ntd, residues 21-324 based on pedv cv777) and the c-terminal domain (s1 ctd, residues 253-638). however, the cellular receptor of pedv is still unknown (li et al., 2017b; shirato et al., 2016) . although the sialic acid binding activity of s1-ntd was observed in many coronaviuses and facilitated virus replication (schwegmann-wessels et al., 2011; li et al., 2016; desmarets et al., 2014) , s1-ntd is dispensable for some mutants of transmissible gastroenteritis coronavirus (tgev) and pedv (schwegmann-wessels et al., 2011; oka et al., 2014; suzuki et al., 2016; hou et al., 2017) . pedv variants with a big deletion in the s1-ntd have been designated as s1 ntd-del pedvs (hou et al., 2017) . they have been detected in field pig samples, indicating sufficient replication of these s1 ntd-del pedvs in vivo (suzuki et al., 2015; diep et al., 2017; zhang et al., 2018a; su et al., 2018) . interestingly, most field s1 ntd-del pedvs exist as a member of coinfection of pigs with the s-intact pedv (diep et al., 2017; su et al., 2018) . 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., 2011) . 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., 2000) . 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., 2004) and human noroviruses (ettayebi et al., 2016) . since intestinal epithelial cells are exposed to bile acids, we hypothesized that bile acids promote pedv infection. previously, we generated two recombinant pedvs, pedv icpc22a and icpc22a-s1δ197 (hou et al., 2017) . pedv icpc22a is an infectious cdna clone-derived virus of the emerging highly virulent pedv pc22a strain. on the other hand, pedv icpc22a-s1δ197 is a mutant version of icpc22a bearing a 197-aa deletion (residues 34-230) in the s1 ntd region, corresponding to the s1 domain 0 (s1°) of the spike protein (li et al., 2017a; hou et al., 2017) . 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., 2016) . ipec-dq cell is a subclone of porcine small intestinal cell line ipec-j2 (zhang et al., 2018b) . 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 ccl81) were cultured in dulbecco's modified eagle's medium (dmem, gibco, carlsbad, ca, usa) supplemented with 5% fetal bovine serum (fbs, hyclone, logan, ut, usa) and 1% penicillin/streptomycin (gibco). for virus propagation in vero cells, the maintenance medium was dmem supplemented with 0.3% tryptose phosphate broth solution (tpb, sigma, st. louis, mo, usa), 1% penicillin/streptomycin (gibco) and 10 μg/ml trypsin (2.5% 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-j2 cells, a porcine small intestinal cell line, and maintained in roswell park memorial institute 1640 medium (rpmi-1640, gibco) supplemented with 10% fbs and 1% penicillin/streptomycin (zhang et al., 2018b) . for virus propagation in ipec-dq cells, maintenance medium was rpmi-1640 containing 0.3% tpb, 1% penicillin/streptomycin and 1 μg/ml trypsin. two recombinant pedv, icpc22a and icpc22a-s1δ197, were generated previously by our lab using the infectious clone of pedv pc22a strain (hou et al., 2017) . 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) 56h4, 60f11 and 72 a8 targeting the s1°domain of pedv s protein (fig. s1a ) was provided by dr. berend jan bosch, utrecht university, utrecht, netherlands (li et al., 2017a) . the mab sd6-29 targeting pedv nucleocapsid (n) protein was provided by drs. eric nelson and steven lawson, south dakota state university. the guinea pig polyclonal antibody (pab) gp17 targeting the s1 subunit of the s protein of pedv pc177 strain (s1δ197) (fig. s1a) , rabbit pab rb9 targeting pedv n protein and swine pedv-positive serum (virus neutralization titer 1:1024) were generated by our laboratory previously (hou et al., 2019) . 2.2. duplex rt-qpcr method for the detection and differentiation of pedv icpc22a and icpc22a-s1δ197 based on the nucleotide sequence differences in the s1 ntd coding region between the icpc22 a and icpc22 a-s1δ197, a duplex taqman real-time reverse-transcription quantitative pcr (rt-qpcr) was developed. we designed the primers and probes (table 1 , fig. s1b ) 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-20320f and pedv-21816r (oka et al., 2014) was used to amplify a 1.5 kb or a 0.9 kb fragment covering pedv partial nsp16 gene and s1 gene of icpc22 a and icpc22 a-s1δ197, respectively. the pcr products were purified using gel extraction kit (qiagen) and used as standard dna. the reaction consisted with 4 μl 5 × pcr buffer, 0.8 μl 10 mm dntps (qiagen), 1.2 μl each primer (10 μm), 0.4 μl each probe (5 μm), 0.2 μl rnasin (promega, wi, usa), 0.8 μl enzyme mix (qiagen), 1.6 μl each dna/rna and 6.6 μl sterilized water. the rt-qpcr procedure was: 50°c for 30 min, 95°c for 15 min and 45 cycles of 95°c for 15 s and 60°c for 60 s. we generated two standard curves using light-cycler® 96 system (roche life science, indianapolis, in, usa). the two standard curves for the serially diluted dna fragments of icpc22a and icpc22a-s1δ197 showed regressions with coefficients of -0.2873 and -3.009, respectively, and the corresponding correlation coefficient (r 2 ) of 0.9987 and 0.9963, 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 icpc22 a and icpc22 a-s1δ197 using serially diluted, cell cultured viruses (table s1 and s2). for the samples of mixed viruses, the duplex rt-qpcr was performed to differentiate the s gene of each virus. the detection limit was 5 log 10 s gene copies/ml for both icpc22 a and icpc22 a-s1δ197. the gn piglets were derived and raised as described previously (saif et al., 1996) . thirty-one gn piglets were assigned to five experimental groups: (1) icpc22 a (n = 14; 100 pfu/pig); (2) icpc22a-s1δ197 (n = 7; 100 pfu/pig); (3) coinfection of icpc22a and icpc22 a-s1δ197 (n = 7; 100 pfu/pig of each virus); (4) mock (n = 2; pbs); (5) second passage of coinfection [n = 1; small intestinal contents (sic) (sample #pe1158) of one piglet in group 3 collected at 2 day post inoculation (dpi), at a dose of 10 log 10 copies/ml based on the n gene, 9 log 10 copies/ml and 7 log 10 copies/ml based on the icpc22a-specific s gene and icpc22a-s1δ197-specific s gene, respectively, corresponding to 4 table 1 primers and probes of the duplex rt-qpcr. log 10 pfu/ml of infectious pedv] . at 5 days of age, piglets in groups 1-4 were orally inoculated with individual or the mixture of the viruses. the group 5 pig was inoculated at 12 days of age and was euthanized at 1 dpi. to examine histopathological changes, one or two piglets of groups 1-4 were euthanized at 0.5 dpi and 2-3 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: 0, solid; 1, pasty; 2, semiliquid (mild diarrhea); and 3, liquid (severe diarrhea). total fecal rna was extracted using magmax-96 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 96-well plates and determined as 50% tissue culture infective doses (tcid 50 ) (reed and muench, 1938) . at necropsy, three sections of the jejunum (proximal, middle, and distal) and one section of the ileum were collected and fixed in 3.7% 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 sd6-29 as the primary antibody (hou et al., 2017) . 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-70 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, 10 villi and crypts were measured. to detect and differentiate icpc22 a-and icpc22 a-s1δ197-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 56h4, 60f11 and 72a8 (1: 100 in pbs) (li et al., 2017a) targeting the s1°domain of pedv s protein was used as the primary antibody for the staining of icpc22 a-infected cells only. tissues were incubated at 4℃ overnight, after three washings with pbst (pbs with 0.1% tween20), alexa fluor 594-conjugated goat antimouse igg (h + l) (thermo fisher scientific) (1: 500 diluted in pbs) was used as the second antibody and tissues were incubated at room temperature for 2 h. after washing with pbst, guinea pig pab gp17 (1: 200 diluted in pbs), targeting the s1δ197 and was for the staining of both viruses, was added. then, the slides were incubated at room temperature for 2 h. after washing three times with pbst, alexa fluor 488-conjugated goat anti-guinea pig igg (h + l) (thermo fisher scientific) (1: 500 diluted in pbs) was used as the 2nd antibody and the slides were incubated at room temperature for 2 h. after washing with pbst, 1 mg/ml dapi (4′,6-diamidino-2-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-70 fluorescent microscope. red, green and blue fluorescence staining was merged by imagej software (https://imagej.nih. gov). 2.5. isolation of the pig-passaged icpc22 a and icpc22 a-s1δ197 from the sic of a pig coinfected with the viruses the sic (sample #pe1158) of a coinfected piglet in group 3 was positive for both viruses by the duplex rt-qpcr. virus isolation was performed using plague assays (oka et al., 2014) . the sic was diluted in dmem and the 10% suspension was vortexed briefly followed by centrifugation at 15,000 ×g for 3 min at 4℃. a series of 10-fold dilutions (10 −1 -10 −5 ) of the supernatants were prepared in dmem (containing 7.5 μg/ml trypsin) and were used immediately for inoculation of vero cell monolayers in 6-well plates (200 μl per well). after incubating at 37℃ for 1 h, the inoculum was removed and the monolayers were washed once with pbs. then 2 ml/well of agarose overlay was added. the plate was kept for 3 days before picking up clones. for propagating each clone, vero cell monolayers in 96-well plates were prepared and individual plagues were picked and added into individual wells. at 3 dpi, rna was extracted and the duplex rt-qpcr method specific for both viruses was performed. 2.6. multi-step growth kinetics and if staining of coinfection of pedv icpc22a and icpc22a-s1δ197 in vero and ipec-dq cells the growth kinetics of icpc22a-s1δ197 and icpc22a during the coinfection of the two viruses were investigated in vero and ipec-dq cells. cell monolayers on 96-well plates were inoculated with one of the three inocula: (1) 0.01 multiplicity of infection (moi) of icpc22a; (2) 0.01 moi of icpc22a-s1δ197; and (3) 0.01 moi of icpc22a and 0.01 moi of icpc22 a-s1δ197. viruses were diluted in dmem containing 10 μg/ml trypsin and were added to cell monolayers. after incubating at 37℃ for 2 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 37℃. the samples were collected at different time points (2 h, 8 h, 12 h, 36 h, 48 h) and frozen (-80℃) and thawed once before testing. rna was extracted using the magmax-96 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 8 h post infection (hpi), the procedure in 2.4 section was performed. 2.7. infection of pedv icpc22a and icpc22a-s1δ197 in na-treated vero and ipec-dq cells vero/ipec-dq cell monolayers in 96-well plates were treated with 250 mu na in dmem/rpmi-1640 or mock dmem/rpmi-1640 at 37℃ for 2 h. after three washings with dmem/rpmi-1640, cells were inoculated with icpc22 a or icpc22 a-s1δ197 at a moi of 0.02 with 10 μg/ml trypsin for 2 h. next, the inoculum was removed followed by three washings. dmem/rpmi-1640 containing 50 μg/ml soybean trypsin inhibitor (sbti) and swine pedv positive serum (virus neutralization titer 1:1024, 1:500 dilution) was added to prevent virus spreading. cells were fixed with acetone-methanol (1:4) at 6 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., 2017) . briefly, rabbit pab rb9 (1:1000) was used as the primary antibody. after incubating at 4℃ overnight, cells were washed three times. then alexa fluor 488-conjugated goat anti-rabbit igg (h + l) (thermo fisher scientific) (1:1000) was used as the 2 nd antibody and the cells were incubated at room temperature for 1 h. after washing and adding mounting medium, cells were observed and picture were captured by using olympus ix-70 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 100%. 2.8. effect of mucin, bile and bile acids on the infection of pedv icpc22a and icpc22a-s1δ197 in vero and ipec-dq cells viruses (icpc22a or icpc22a-s1δ197) were mixed with different concentrations of bm (0, 0.1, 0.3, 0.5 mg/ml) or pgm (0, 0.5, 1.0, 2.5, 5.0 mg/ml). then 10 μg/ml trypsin were added to the mixture. monolayer of vero/ipec-dq cells in 96-well plates were inoculated with each virus mixture at a moi of 0.02. the plates were incubated at 37℃ for 2 h, then the inoculum was removed. after three washings, dmem/rpmi-1640 containing 50 μg/ml sbti and swine pedv positive serum (1:500 dilution) was added. cells were fixed at 6 hpi. the numbers of ffu were determined by if assay for the staining of pedv n proteins (see section 2.7). the percentages of ffu were calculated by the value of mock group as 100%. similarly, different concentration of swine bile (0, 0.1, 0.3, 0.5, 1.0%) and bile acids (gcdca, cdca, dca, and udca) at 0, 25, 50, 100, 200 μm were tested. statistical analysis was performed using graphpad prism 7 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 < 0.05 was considered as significant. 3.1. the replication of icpc22a was eventually enhanced during coinfection with icpc22a-s1δ197 in neonatal gn piglets as shown in fig. 1 , 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 1.5 dpi, which was delayed half a day compared with the icpc22a group (1 dpi) but earlier than the icpc22a-s1δ197 group (> 4 dpi). by 4 dpi, all pigs in the icpc22a and coinfection groups but no pigs in the icpc22a-s1δ197 group died or showed moribund. compared with the peak fecal pedv n gene shedding titer (11.6 ± 0.2 log 10 copies/ml) of piglets in the icpc22a group (1 dpi), pigs in the coinfection group had a significantly higher peak titer (13.6 ± 0.7 log 10 copies/ml) ( fig. 1b and table 2 ) at a delayed time point (1.5 dpi). in addition, the peak fecal infectious pedv shedding titer of the coinfection group (5.8 ± 0.4 log 10 tcid 50 /ml) were significantly higher than that of the single infection groups (4.9 ± 0.1 and 1.8 ± 1.8 log 10 tcid 50 /ml of icpc22 a and icpc22 a-s1δ197 groups, respectively) ( table 2) . interestingly, the peak s gene shedding titers of icpc22 a (11.4 ± 0.5 log 10 copies/ml) in the coinfection group at 1.5 dpi was significantly higher than that of icpc22 a group (10.5 ± 0.5 log 10 copies/ml) at 1 dpi (fig. 1c, table 2 ). however, the peak s gene shedding titers (5.8 ± 1.2 log 10 copies/ml) of icpc22 a-s1δ197 in the coinfection group was significantly lower than that of the icpc22 a-s1δ197 group (7.6 ± 1.7 log 10 copies/ml) (fig. 1c , table 2 ). in the second passage of coinfection, only icpc22 a but not icpc22 a-s1δ197 s gene was detected in the large intestinal contents (lic) of the piglet, which was orally inoculated with the sic (sample #pe1158) of one pig in the coinfection group and was positive for both viruses. the lic had a virus titer of 8.8 log 10 tcid 50 /ml for infectious pedv, 12.3 log 10 s gene copies/ml and 14.6 log 10 n gene copies/ml for icpc22a. histopathological examination was performed and villous atrophy was observed in the jejunum and ileum of piglets in all pedv-inoculated groups ( fig. 2a and b ). the small intestinal villi in the icpc22a-s1δ197 group had a milder villous atrophy than those in the icpc22a 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. 2b) . the ratios of the icpc22 a and coinfection groups were similar and were significantly lower than that of the icpc22 a-s1δ197 group. ihc staining showed that no pedv-positive intestinal epithelial cells were observed in all pedv-inoculated pigs fig. 1 . evaluation of the replication of icpc22a and icpc22a-s1δ197 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: 0, solid (normal); 1, pasty (normal); 2, semiliquid (mild diarrhea); and 3, liquid (severe diarrhea). two piglets from icpc22a, icpc22a-s1δ197 and coinfection groups, and one pig from mock group were euthanized at 12 hpi and 2-3 dpi, respectively, for histopathological examination. (b) fecal pedv n gene rna shedding titers (for both icpc22a and icpc22a-s1δ197) 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 0.5 dpi (data not shown). at 2-3 dpi, a few pedv n antigens were observed in the small intestine of the icpc22a-s1δ197-infected piglets, while extensive antigens were stained in those of the icpc22a and coinfection piglets ( fig. 2a) . by if staining for the pedv s1 proteins, a few pedv antigens were observed in the jejunum of the icpc22a-s1δ197-inoculated piglets (green alone, positive for s1δ197 only), while many antigens were observed in those of the icpc22 a-inoculated piglets [yellow, representing positive for both s1δ197 (green) and s1°d omain (red)] and the coinfection piglets (yellow) (fig. 2c) . 3.2. icpc22a alone, but not icpc22a-s1δ197 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 72 plagues and found that 55 plagues were positive for icpc22a and 17 plagues were positive for both viruses by the duplex rt-qpcr. no plagues were positive for s1 ntd-del pedv alone. during coinfection in vero cells, icpc22a replicated to a titer of 10.10 ± 0.05 log 10 copies/ml at 72 hpi, which was similar to that of the single infection (10.20 ± 0.09 log 10 copies/ml) (fig. 3a) . however, icpc22a-s1δ197 s gene peak titer was 9.80 ± 0.03 log 10 copies/ ml, which was significantly lower than that of the single infection (10.30 ± 0.04 log 10 copies/ml). in vero cells, icpc22a alone and icpc22a-s1δ197 alone replicated to similar titers. similarly, during coinfection in ipec-dq cells, icpc22a replicated to a similar titer to its single infection (8.60 ± 0.18 vs. 8.70 ± 0.11 log 10 copies/ml at 48 hpi (fig. 3c ). icpc22a-s1δ197 s gene peak titer was 7.03 ± 0.12 log 10 copies/ml, which was significantly lower than that of the single infection (7.49 ± 0.25 log 10 copies/ml). in contrast to those results in vero cells, icpc22a alone replicated to significantly higher titers than icpc22a-s1δ197 alone in ipec-dq cells. by if staining, more icpc22a-s1δ197-infected cells (green) were observed in vero cells than in iepc-dq cells in both icpc22a-s1δ197 alone and co-infection conditions ( fig. 3b and d) . at the second passage of the coinfection, icpc22 a rna and antigens were detected in both cells; however, icpc22 a-s1δ197 rna and antigens were detected exclusively in vero cells but not in ipec-dq cells (data not shown). 3.4. the percentage of infectivity of icpc22a was significantly lower than that of icpc22a-s1δ197 in na-treated vero and ipec-dq cells after removing sialic acids from the cell surface using 250 mu na in vero and ipec-dq cells, the percentages of infectivity of icpc22a and icpc22a-s1δ197 were reduced significantly (fig. 4) . however, in both cells treated with na, the percentages of infectivity of icpc22a were significantly lower than those of icpc22a-s1δ197. 3.5. mucin enhanced the infectivity of icpc22a but had no or inhibition effects on icpc22a-s1δ197 in both vero and ipec-dq cells in vero cells, 0.1-0.3, 0.5 and 1.0 mg/ml bm increased, had no effects and decreased the infectivity of icpc22a, respectively (fig. 5a ). 0.5-5.0 mg/ml pgm increased the infectivity of icpc22a 1.7-2.5 fold (fig. 5b) . however, the infectivity of icpc22a-s1δ197 was unaffected or inhibited significantly by bm and pgm at the tested concentration ( fig. 5a and 5b) . in ipec-dq cells, the infectivity of icpc22a was increased 1.7-2.6 fold and 2.6-3.8 fold by bm (0.1-1.0 mg/ml) and pgm (0.5-2.5 mg/ ml), respectively. however, there was no effects at the highest pgm concentration tested (5.0 mg/ml). on the other hand, low to medium concentrations of bm (0.1-0.5 mg/ml) and pgm (0.5-1.0 mg/ml) had no effects on the infectivity of icpc22 a-s1δ197. only high concentrations of bm (1.0 mg/ml) and pgm (2.5-5.0 mg/ml) reduced the infectivity of icpc22 a-s1δ197 significantly ( fig. 5c and 5d ). 3.6. bile and bile acids enhanced the infectivity of icpc22a but had no or inhibition effects on icpc22a-s1δ197 in vero cells, low to medium concentrations of swine bile (0.1-0.5%) increased 2.4-3.3 fold of the infectivity of icpc22a (fig. 6a) . (fig. 6b-e) . however, the infectivity of icpc22a-s1δ197 was unaffected (0.1-0.3%) or inhibited significantly by bile (0.5-1.0%) by bile (fig. 6a) . bile acids a piglets in group 1 were inoculated with 100 pfu/pig of icpc22 a; piglets in group 2 were inoculated with 100 pfu/pig of icpc22a-s1δ197; piglets in group 3 were inoculated with 100 pfu/pig of each virus; piglets in group 4 were inoculated with pbs; piglets in group 5 were inoculated with the sic of piglet in group 3 with a dose of 4 log 10 pfu/ml targeting n gene of pedv. b fecal consistency (fc) was scored as follows: 0, solid, 1, pasty, 2, semiliquid; and 3, liquid. an fc score of 2 and 3 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 icpc22a or icpc22 a-s1δ197 is 5 log 10 copies/ml. e the detection limit of pedv rt-qpcr targeting the n gene of both icpc22a and icpc22 a-s1δ197 is 4.8 log 10 copies/ml . f the pig number for diarrhea observation is 1 or 2 less than the total pig number because 1 or 2 pigs of each group were euthanized at 12 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 < 0.05). '-', not detected. y. su et al. veterinary microbiology 228 (2019) 202-212 at the tested concentrations had no effects on the infection of icpc22 a-s1δ197 (fig. 6b-e) . similarly, in ipec-dq cells, the infectivity of icpc22a was increased significantly by 0.1-0.3% bile but was decreased significantly by the concentration of 1.0% (fig. 6f) . however, 0.1-1.0% bile decreased the infectivity of icpc22a-s1δ197 significantly. bile acids gcdca (fig. 6g-j) . no significant effects of bile acids on icpc22a-s1δ197 were observed except for that the highest concentration (200 μm) of gcdca and dca decreased its infectivity significantly (fig. 6g-j) . fig. 2 . histopathological examination of the gn piglets infected with individual or both icpc22a and icpc22a-s1δ197. (a) ihc staining of pedv n proteins in the jejunal and ileal sections of piglets that died or were euthanized at 2-3 dpi (magnification, 200×) . 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 2-3 dpi. for each intestinal section, 10 villi and crypts were measured. data are shown as the m ± sd. number of asterisks indicate significant difference between groups (*, p < 0.05; **, p < 0.01; ***, p < 0.001). 'ns', not significant. (c) if staining of pedv s proteins in the jejunal sections of piglets that died or were euthanized at 2-3 dpi (magnification, 200 x). tissue sections were stained for the detection of icpc22a and icpc22a-s1δ197 antigens targeting the s1δ197 protein (green), and for icpc22a antigens targeting the s1°domain (red), and counterstained for cell nuclei (blue). in the merged images, yellow dots represented icpc22a-infected (or co-infected in coinfection group) cells (merged from red and green) and the green dots represented icpc22a-s1δ197 infection alone. (magnification, 200 x) . cells were stained for the detection of icpc22a and icpc22 a-s1δ197 antigens targeting the s1δ197 protein (green), and for icpc22a antigens targeting the s1°domain (red), and counterstained for cell nuclei (blue). in the merged images, yellow dots represented icpc22a-infected (or co-infected in coinfection condition) cells and the green dots represented icpc22a-s1δ197 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 < 0.05). previously, our laboratory isolated the first s1 ntd-del pedv strain, pc177, from vero cell culture (oka et al., 2014) . recent studies revealed that s1 ntd-del pedvs naturally evolved in the field and often coinfected pigs with the s-intact pedv (suzuki et al., 2015; diep et al., 2017; zhang et al., 2018a; su et al., 2018) . the s1 ntd-del pedv had no tissue tropism change compared with the s-intact pedv (suzuki et al., 2016; hou et al., 2017) . this differs from porcine respiratory coronavirus (prcv) that is a s1 ntd-del version of tgev and changes the major tissue tropism from intestines to respiratory tract (zhang et al., 2007) . in this study, we investigated the replication of s1 ntdfig. 4 . infection of pedv icpc22a and icpc22a-s1δ197 in neuraminidase (na)-treated vero and ipec-dq cells. vero or ipec-dq cell monolayers in 96-well plates were treated with 250 mu na or mock at 37℃ for 2 h. after three washings with dmem/rpmi-1640, cells were inoculated with equal amounts of virus at a moi of 0.02 diluted in the medium containing 10 μg/ml trypsin. after incubation at 37℃ for 2 h, the inoculum was removed followed by three washings. dmem/rpmi-1640 containing 50 μg/ml soybean trypsin inhibitor (sbti) and swine pedv positive serum (virus neutralization titer 1:1024, 1:500 dilution) was added. at 8 hpi, cells were fixed with acetone-methanol (1:4) 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 < 0.05; **, p < 0.01; ***, p < 0.001). fig. 5 . effect of bovine mucin (bm) and porcine gastric mucin (pgm) on the infection of pedv icpc22a and icpc22a-s1δ197 in vero (a and b) and ipec-dq cells (c and d). monolayers of vero or ipec-dq cells in 96-well plates were inoculated with a moi of 0.02 icpc22a or icpc22a-s1δ197 in medium containing 10 μg/ml trypsin and different concentrations of bm or pgm. after incubation at 37℃ for 2 h, the inoculum was removed followed by three washings. dmem/rpmi-1640 containing 50 μg/ml soybean trypsin inhibitor (sbti) and swine pedv positive serum (virus neutralization titer 1:1024, 1:500 dilution) was added. at 8 hpi, cells were fixed with acetone-methanol (1:4) 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 < 0.05; **, p < 0.01; ***, p < 0.001). 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 icpc22 a and icpc22 a-s1δ197 (table s1) although it had a similar detection limit (5 log 10 copies/ml) for both viruses. for example, 9 log 10 s gene copies/ ml corresponded to 6.2 and 4.2 log 10 ffu/ml for icpc22a and icpc22a-s1δ197, respectively. it may reflect the fact that icpc22a-s1δ197 replicates to lower peak infectious titers (∼5.0 log 10 ffu/ml) than that (∼7.0 log 10 ffu/ml) of icpc22a in vero cells although both viruses replicated to similar peak titers based on the s gene (∼10.0 log 10 copies/ml) (fig. 3a) . in the mixture of two viruses, the detection fig. 6 . effect of bile and bile acids on the infection of pedv icpc22a and icpc22a-s1δ197 in vero (a-e) and ipec-dq cells (f-j). monolayers of vero or ipec-dq cells in 96-well plates were inoculated with a moi of 0.02 icpc22 a or icpc22 a-s1δ197 in medium containing 10 μg/ml trypsin and different concentrations of bile or bile acids. after incubation at 37℃ for 2 h, the inoculum was removed followed by three washings. dmem/rpmi-1640 containing 50 μg/ml sbti and swine pedv positive serum (virus neutralization titer 1:1024, 1:500 dilution) was added. at 8 hpi, cells were fixed with acetone-methanol (1:4) 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 < 0.05; **, p < 0.01; ***, p < 0.001). sensitivity of each virus differed significantly (tables s2). for the detection of icpc22 a at high titers (6.2 log 10 ffu/ml) and as the predominant virus in the mixture of two viruses (icpc22a: icpc22 a-s1δ197 = 10 2 -10 5 :1), the s gene titers (∼8.8-9.1 log 10 s gene copies/ ml) (table s2 ) were similar to that (∼9.0 log 10 s gene copies/ml) in the single virus alone (table s1 ). because icpc22 a replicated to high titers and was the predominant virus during the coinfection in pigs, we can predict that the peak infectious titer of icpc22a in coinfection was ∼1.0 log 10 -higher than that in its single infection based on the duplex rt-qpcr results (∼11.4 vs ∼10.5 log 10 s gene copies/ml) (table 2) . so, we conclude that the replication of icpc22a was interfered at the beginning based on delayed timing (1.5 vs 1.0 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 icpc22a-s1δ197 decreased significantly and icpc22a-s1δ197 became undetectable when icpc22a was high (6.2 log 10 ffu/ml) and icpc22 a-s1δ197 infectious titers were lower than 3.2 log 10 ffu/ml in the mixture of two viruses (table s2 ). therefore, the decreased peak s gene titer (∼5.8 log 10 copies/ml) of icpc22a-s1δ197 in coinfection of pigs compared with that (∼7.6 log 10 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 icpc22a-s1δ197 during coinfection (table 2 ). in the first passage of coinfection, we observed much lower numbers of s1 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, s1 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 s1 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 s1 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., 2017; su et al., 2018) . s1 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, s1 ntd-del pedv rna was detected in vero cells but not in ipec-dq cells, probably due to the lower replication efficiency of the s1 ntd-del pedv in ipec-dq cells than in vero cells ( fig. 3a and c) . similar to other sialic acid-binding coronaviruses, the s1 ntd of pedv has a sialic acid binding activity, and the sugar-binding activities of a field isolate pedv variant chgd-01 was stronger than that of the prototype strain pedv cv777 using bm (deng et al., 2016) . the sialic acid binding activity occurred within the n-terminal 249 residues and the capacity of sialic acid binding differs among pedv strains using an hemagglutination assay . the recombinant virus used in this study, pedv icpc22 a-s1δ197, is a s1 ntd-del version of icpc22a. it lost most sialic acid binding activity tested in vero cells (hou et al., 2017) . in this study, we comparatively tested icpc22 a-s1δ197 and icpc22 a in vero and ipec-dq cells treated with na. similar to the previous reports, our results confirmed that the binding of s1 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 5-n-acetyl-9-o-acetylneuraminic acid (neu5,9ac2), 5-n-glycolylneuraminic acid (neu5gc), and 5-n-acetylneuraminic acid (neu5ac). among them, neu5gc and neu5ac can serve as receptors or co-receptors for some alphacoronaviruses (e.g., tgev) and gammacoronaviruses [e.g., infectious bronchitis virus (ibv)] (cavanagh and davis, 1986; krempl et al., 1997) . similar to tgev, which uses neu5gc and neu5 ac as co-receptors (krempl et al., 1997; schultze et al., 1996; schwegmann-wessels and herrler, 2006) , the s1 ntd of pedv interacted with sugar (deng et al., 2016) . pgm contains 0.5-1.5% 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 s1 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 s1 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 s1 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 1.0 mg/ml bm effects on s-intact pedv in vero cells (fig. 5a) . the major components of bile include bile acids, cholesterol, lipids, bilirubin, proteins and carbohydrates. the bile acids are stored in the gall bladder (at ∼300 mm) and released into the small intestine (mcleod and wiggins, 1968) . in the small intestine, the total bile acids have a concentration of 2-30 mm (dowling, 1973) . 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, 1985) . 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 s1 ntd-del pedv. these results suggest that the bile-and bile acid-mediated promotion of pedv infection is related to the s1 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 (< p10) (kim et al., 2017) . 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 s1 ntd-del pedv to outcompete the s-intact pedv may account for the fact that s1 ntd-del pedvs were exclusively detected from coinfection with the s-intact pedv (diep et al., 2017; su et al., 2018) . 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., 1994; brim et al., 1995) . prcv functions as a natural effective vaccine against tgev. however, for pedv, the clinically mild s1 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 100% mortality rates of piglets in the icpc22 a-infected pigs (hou et al., 2017) and coinfection groups, we currently cannot determine whether the pathogenicity of pedv icpc22 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, 2018) . in addition, the emergence of s1 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 s1 ntd-del pedv in pigs. we found that mucin, bile and bile acids can all increase the infection of sintact pedv but not the s1 ntd-del pedv. this feature may help explain why s-intact pedv outcompetes s1 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 protocols approved by institutional animal care and use committee (iacuc) of the ohio state university (osu). mechanism of bile acid biosynthesis in mammalian liver cellular immune responses of pigs after primary inoculation with porcine respiratory coronavirus or transmissible gastroenteritis virus and challenge with transmissible gastroenteritis virus coronavirus ibv: removal of spike glycopolypeptide s1 by urea abolishes infectivity and haemagglutination but not attachment to cells bile acids are 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interest. none. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.vetmic.2018.11.025. key: cord-254916-y1rw9q11 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-2 replication in vero e6 cells: replication kinetics, rapid adaptation and cytopathology date: 2020-06-22 journal: j gen virol doi: 10.1099/jgv.0.001453 sha: doc_id: 254916 cord_uid: y1rw9q11 the sudden emergence of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) at the end of 2019 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-2 and sars-cov and analysed the cytopathology caused by the two closely related viruses in the commonly used vero e6 cell line. compared to sars-cov, sars-cov-2 generated higher levels of intracellular viral rna, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium. immunofluorescence microscopy of sars-cov-2-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-2 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 e6 cells – sars-cov-2 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 s1 and s2 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 2 (sars-cov-2) [1] 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 [2] , 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 1960s, annually causing a substantial number of common colds [3, 4] . 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) [5, 6] in [2002] [2003] , middle east respiratory syndrome-coronavirus (mers-cov) [7, 8] since 2012 (and probably earlier) and the current pandemic sars-cov-2 [9, 10] . the latter agent emerged near wuhan (pr china) in the fall of 2019 and its animal source is currently under investigation [11] [12] [13] . transmission to humans of sars-cov and mers-cov was attributed to civet cats [14] and dromedary camels [15] , 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 [2, [16] [17] [18] [19] . the genetic diversity of bat covs and their phylogenetic relationships with the four known endemic hcovs (oc43, hku1, 229e and nl63; 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 [20] . the potential of multiple covs from different genera to cross species barriers had been predicted and documented previously [2, 16-19, 21, 22] , 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 26 to 34 kilobases [23] . the cov genome is single-stranded and its 5′-proximal two-thirds encode for the large and partially overlapping replicase polyproteins pp1a and pp1ab (4000-4500 and 6700-7200 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 16 cleavage products (non-structural proteins, nsps) by two internal proteases, the papain-like protease (pl pro ) in nsp3 and the 3c-like or 'main' protease (m pro ) in nsp5 [24] . specific transmembrane nsps (nsp3, 4 and 6) then cooperate to transform intracellular membranes into a viral replication organelle (ro) [25] 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 3′-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 [26] . 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 [27] . the latter process depends on a set of enzymatic activities [24] including an rna-dependent rna polymerase (rdrp; in nsp12), rna helicase (in nsp13), two methyltransferases involved in mrna capping (a guanine-n7-methyltransferase in nsp14 and a nucleoside-2′-o-methyltransferase in nsp16) and a unique exoribonuclease (exon, in nsp14) that promotes the fidelity of the replication of the large cov genome [28] . 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 (nsp7-10) that mainly appear to serve as cofactors/modulators of other nsps. the newly emerged sars-cov-2 was rapidly identified as a cov that is relatively closely related to the 2003 sars-cov [9, 29, 30] . the two genome sequences are about ~80 % identical and the organization of orfs is essentially the same. the overall level of amino acid sequence identity of viral proteins ranges from about 65 % in the least conserved parts of the s protein to about 95 % 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 2003 sars-cov [1] . the close phylogenetic relationship also implies that much of our knowledge of sars-cov molecular biology, accumulated over the past 17 years, can probably be translated to sars-cov-2. many reports posted over the past months have described such similarities, including the common affinity of the two viruses for the angiotensinconverting enzyme 2 (ace2) receptor [9, 31] . this receptor is abundantly expressed in vero cells (african green monkey kidney cells). since 2003, 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-2 replication in vero cells and compare it to the frankfurt-1 sars-cov isolate from 2003 [32, 33] . when requesting virus isolates (february 2020), 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 1 week) with their first sars-cov-2 isolate (originally named 2019-ncov/victoria/1/2020 and subsequently renamed betacov/australia/vic01/2020 [34] , which will be used throughout this study. until now, this isolate has been provided to 17 other laboratories worldwide to promote the rapid characterization of sars-cov-2, 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-2 in vero e6 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 13 available sars-cov-specific antisera (recognizing ten different viral proteins) with their sars-cov-2 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-2 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-2 s protein that contains the so-called furin-like cleavage site. vero e6 cells and huh7 cells were grown as described previously [35] . sars-cov-2 isolate australia/vic01/2020 (genbank id: mt007544.1 [34] ) 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 e6 cells to obtain a working stock (p2 stock) that was used in all experiments. sars-cov isolate frankfurt 1 [36] was used to compare growth kinetics and other features with sars-cov-2. infection of vero e6 cells was carried out in pbs containing 50 µg ml −1 deaedextran and 2 % fcs (bodinco). the inoculum was added to the cells for 1 h at 37 °c, after which cells were washed twice with pbs and maintained in eagle's minimal essential medium (emem; lonza) with 2 % fcs, 2 mm l-glutamine (paa) and antibiotics (sigma). viral titres were determined by plaque assay in vero e6 cells as described previously [37] . for plaque picking, plaque assays were performed using our p1 stock, while using an overlay containing 1 % of agarose instead of avicel (rc-581; fmc biopolymer). following neutral red staining, small and large plaques were picked and used to inoculate a 10 cm 2 dish of vero e6 cells containing 2 ml of emem-2%fcs medium, yielding p1 virus. after 48 h, 200 µl of the culture supernatant was used to infect the next dish of cells (p2), a step that was repeated one more time to obtain p3 virus. all work with live sars-cov and sars-cov-2 was performed in biosafety laboratory level 3 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 1.5 % agarose gel containing 2.2 m formaldehyde, which was run overnight at low voltage in mops buffer [10 mm mops (sodium salt) (ph 7), 5 mm sodium acetate, 1 mm edta]. dried agarose gels were used for direct detection of viral mrnas by hybridization with a 32 p-labelled oligonucleotide probe (5′-cacatggggatagcactac-3′) that is complementary to a fully conserved sequence located 30 nucleotides upstream of the 3' end of the genome as well as all subgenomic mrnas produced by sars-cov-2 and sars-cov. after hybridization, rna bands were visualized and quantified by phosphorimaging using a typhoon-9410 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 32 p-labelled oligonucleotide probe recognizing 18s ribosomal rna (5′-gatc cgag ggcc tcac taaac-3′). protein lysates were obtained by lysing infected cell monolayers in 4×laemmli sample buffer and were analysed by semi-dry western blotting onto hybond 0.2 µm polyvinylidene difluoride (pvdf) membrane (ge healthcare). membranes were incubated with rabbit antisera diluted in pbs with 0.05 % tween-20 containing 5 % 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 q9 alliance imager (uvitec cambridge). sars-cov-2 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 (25-100 ng) was mixed with random oligonucleotide primers using the nebnext first strand synthesis module kit for illumina (neb) and incubated for 10 min at 94 °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 6000 sequencing system (illumina). subsequently, sequencing reads were screened for the presence of human (grch37.75), mouse (grcm38.p4), e. coli mg1655 (embl u00096.2), phix (refseq nc_001422.1) and common vector sequences (univec and chlsab1.1). prior to alignment, reads were trimmed to remove adapter sequences and filtered for sequence quality. the remaining reads were mapped to the sars-cov-2 genbank reference sequence (nc_045512.2 [38] ). data analysis was performed using bowtie 2 [39] . 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/ 628043. only sars-cov-2-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 s1/s2 region of the s protein gene. this was done for the p2 stock and for the four virus samples of the plaque-picking experiment shown in fig. 1a . first, all reads spanning nt 23 576 to 23 665 of the sars-cov-2 genome were selected. next, reads constituting less than 1 % of the total number of selected reads were excluded from further analysis. the remaining number of reads were 3860 (p2 stock), 1924 (s5p1), 2263 (s5p2), 4049 (s5p3) and 3323 (l8p1). 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 s1/s2 region and ranked by frequency of occurrence (fig. 1b) . the sars-cov-specific rabbit or mouse antisera/antibodies used in this study are listed in table 1 . most antisera were described previously (see references in table 1 ), with the exception of three rabbit antisera recognizing sars-cov nsps 8, 9 and 15. these were raised using full-length (his) 6 -tagged table 1 . sars-cov-specific antisera used and their cross-reactivity with corresponding sars-cov-2 targets antigen type antibody type ifa signal* reference nsp3 (dgd7) transmembrane replicase protein, containing pl pro bacterial expression product rabbit polyclonal ++ [48] nsp4 (fgq4) transmembrane replicase protein synthetic peptide rabbit polyclonal ++ [109] nsp5 (due5) m pro bacterial expression product rabbit polyclonal + [48] nsp6 (gbz7) transmembrane replicase protein synthetic peptide rabbit polyclonal − [109] nsp8 (duk4) rna polymerase co-factor bacterial expression product rabbit polyclonal ++ [48] nsp8 ( * ++, strongly positive; +, positive; -, negative. following a plaque assay of the p1 virus stock, small and large plaques were picked and these virus clones were passaged three times in vero e6 cells, while their plaque phenotype was monitored. in contrast to the large plaque viruses (example l8; bottom row), the plaque phenotype of the small plaque viruses (example s5; top row) rapidly evolved within these three passages. (b) evolution/adaptation of the s protein gene during vero e6 passaging. overview of ngs data obtained for the p2 stock, s5p1/p2/p3 and s8p1 in the s1/s2 region of the sars-cov-2 s protein gene that encodes the so-called furin-like cleavage site. the analysis was based on ngs reads spanning nt 23 576 to 23 665 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 (nsp8 and nsp15) or a synthetic peptide (nsp9, aa 4209-4230 of sars-cov pp1a), which were used to immunize new zealand white rabbits as described previously [40, 41] . cross-reactivity of antisera to sars-cov-2 targets was evaluated microscopically by immunofluorescence assay (ifa) and for some antisera (nsp3 and n protein) also by western blot analysis. double-stranded rna was detected using mouse monoclonal antibody j2 from scicons [42] . cells were grown on glass coverslips and infected as described above [43] . at sars-cov-2 isolate betacov/australia/vic01/2020 was received as a stock derived from two consecutive passages in vero/hslam cells [34] . the virus was then propagated two more times at low m.o.i. in vero e6 cells, in which it caused a severe cytopathic effect (cpe). we also attempted propagation in huh7 cells, using the same amount of virus or a tenfold larger inoculum, but did not observe any cytopathology after 72 h (data not shown [38] and other field isolates [29] , isolate betacov/australia/vic01/2020 exhibits >99.9 % sequence identity. in addition to synonymous mutations in the nsp14-coding sequence (u19065 to c) and s protein gene (u22303 to g), orf3a contains a single non-synonymous mutation (g26144 to u). strikingly, the 3′ utr contains a 10 nt deletion (nt 29 750-29 759; cgaucgagug) located 120 nt upstream of the genomic 3′ end, which is not present in other sars-cov-2 isolates described thus far (>670 sars-cov2 sequences present in genbank on 17 april 2020). in about 71 % of the 95 173 p2 ngs reads covering this position, we noticed a g23607 to a mutation encoding an arg682 to gln substitution near the so-called s1/s2 cleavage site of the viral s protein (see discussion), with the other 29 % 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 p1 virus stock (fig. 1a, leftmost well) and picked multiple plaques of each size, which were passaged three times in vero e6 cells while monitoring their plaque phenotype. interestingly, for several of the small-plaque virus clones (like s5; fig. 1a ) we observed rapid conversion to a mixed or large-plaque phenotype during these three passages, while large-plaque virus clones (like l8) stably retained their plaque phenotype (fig. 1a) . ngs analysis of the genome of a large-plaque p1 virus (l8p1) revealed that >99 % of the reads in the s1/s2 cleavage site region contained the g23607 to a mutation described above. no other mutations were detected in the genome, thus clearly linking the arg682 to gln substitution in the s protein to the large-plaque phenotype observed for the l8p1 virus. next, we also analysed the genomes of the p1, p2 and p3 viruses derived from a small-plaque (s5) that was picked. this virus clone retained its small-plaque phenotype during the first passage ( fig. 1a; s5p1 ), but began to yield an increasing proportion of large(r) plaques during subsequent passages. sequencing of s5p2 (fig. 1b) revealed a variety of lowfrequency reads with mutations near the s1/s2 cleavage site motif (aa 681-687; prrar↓sv), with g23607 to a (specifying the arg682 to gln substitution) again being the dominant one (in ~2.1 % of the reads covering nt 23 576 to 23 665 of the genome). at lower frequencies single-nucleotide changes specifying arg682 to trp and arg683 to leu substitutions were also detected. furthermore, a 10 aa deletion (residues 679-688) that erases the s1/s2 cleavage site region was discovered, as well as a 5 aa deletion (residues 675-679) 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 10 aa deletion or the arg682 to gln point mutation (~22 and~12 % 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-2 to the acquisition of mutations in this particular region of the s protein, which apparently provides a strong selective advantage during passaging in vero e6 cells. to our knowledge, a detailed comparison of sars-cov-2 and sars-cov replication kinetics in cell culture has not been reported so far. therefore, we infected vero e6 cells with the sars-cov-2/p2 virus stock at high m.o.i. to analyse viral rna synthesis and the release of infectious viral progeny (fig. 2a) . this experiment was performed using four replicates per time point and for comparison we included the sars-cov frankfurt-1 isolate [36] , which has been used in our laboratory since 2003. during the early stages of infection (until 8 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 50-fold more) than sars-cov-2-infected cells, with both viruses reaching their plateau by about 14 h p.i. as shown in fig. 2b , despite its transition to a mainly large-plaque phenotype, the largest sars-cov-2/p3 plaques were still substantially smaller than those obtained with sars-cov frankfurt-1. 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 32 p-labelled oligonucleotide probe recognizing a fully conserved 19 nt sequence located 30 nt upstream of the 3′ end of both viral genomes (fig. 3a) . this revealed the anticipated presence of the genomic rna and eight subgenomic mrnas, together forming the well-known 5′-and 3′-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. 2a . the relative abundance of the individual rnas was determined using the 12, 14 and 24 h p.i. samples (averages presented in fig. 3b ) and found to be largely similar, with the exception of sars-cov-2 mrnas 7 and 8, which accumulated to about four and twofold higher levels, respectively. strikingly, in spite of the ultimately lower yield of infectious viral progeny, sars-cov-2 rna synthesis was detected earlier and reached an overall level exceeding that of sars-cov. overall, we conclude that in vero e6 cells, sars-cov-2 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. 2a) . to be able to follow virus replication in sars-cov-2-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 [35] , despite the relatively large evolutionary distance between mers-cov and sars-cov. based on the much closer relationship with sars-cov-2, 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 (nsp3, nsp4, nsp5, nsp8, nsp9, nsp13, nsp15) strongly cross-reacted with the corresponding sars-cov-2 target (fig. 4, table 1 ), the exception being a polyclonal nsp6 rabbit antiserum. likewise, both a polyclonal rabbit antiserum and mouse monoclonal antibody recognizing the n protein cross-reacted strongly (fig. 4b , table 1 ). the same was true for a rabbit antiserum raised against a c-terminal peptide of the sars-cov m protein (fig. 4e) . labelling patterns were essentially identical to those previously documented for sars-cov [45, 46] , with nsps accumulating in the perinuclear region of infected cells, where the elaborate membrane structures of the viral ros are formed (fig. 4a, 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 [46, 47] . the n protein signal was diffusely cytosolic (fig. 4b) , whereas the m protein labelling predominantly showed the expected localization to the golgi complex (fig. 4e) , where the protein is known to accumulate [48] . we next used electron microscopy to investigate the ultrastructural changes that sars-cov-2 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. 5) . compared to mock-infected control cells (fig. 5a-b) , various distinct membrane alterations were observed in cells infected with either sars-cov or sars-cov-2 ( fig. 5c-j) . at 6 h p.i., larger regions with membrane alterations were found particularly in cells infected with sars-cov-2 (data not shown), which may align with the somewhat faster onset of intracellular rna synthesis in sars-cov2-infected vero e6 cells (fig. 3a) . from 8 h p.i. onwards, sars-cov-and sars-cov-2-infected cells appeared more similar (fig. 5c-j) . double-membrane vesicles (dmvs) were the most prominent membrane alteration up to this stage (fig. 5d -e, h-i). in addition, convoluted membranes [46] were readily detected in sars-cov-infected cells, while zippered er [25, 49, 50] appeared to be the predominant structure in sars-cov-2-infected cells (fig. 5e , i, white arrowheads). as previously described for sars-cov [46] , sars-cov-2-induced dmvs also appeared to fuse through their outer membrane, giving rise to vesicle packets that increased in numbers as infection progressed (fig. 5f , k, white asterisks). virus budding near the golgi apparatus, presumably into smooth membranes of the er-golgi intermediate compartment (ergic) [45, 51, 52] , was frequently observed at 8 h p.i. (fig. 5k-l, o-p) . this step is followed by transport to the plasma membrane and release of virus particles into extracellular space. by 10 h p.i., released progeny virions were abundantly detected around all infected cells (fig. 5m -n, q-r). interestingly, whereas spikes were clearly present on sars-cov progeny virions, a relatively large proportion of sars-cov-2 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-2 virions. this could potentially reduce the yield of infectious particles and may contribute to the lower progeny titres obtained for this virus (fig. 2a) . in order to establish and validate a cpe-based assay to identify potential inhibitors of sars-cov-2 replication, we selected four previously identified inhibitors of cov replication: remdesivir [53, 54] , chloroquine [55, 56] , alisporivir [57, 58] and pegylated interferon alpha (peg-ifn-α) [35, 59] [110, 111] respectively; fig. 6a ) than previously reported by others, but this may be explained by technical differences like a longer assay incubation time (72 h instead of 48 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 50 ) values of >100 µm, a selectivity index >22.5 was calculated. chloroquine potently blocked virus infection at low-micromolar concentrations, with an ec 50 value of 2.3±1.1 µm for both viruses (cc 50 >100 µm, si>45.5; fig. 6b ). 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 50 values of 4.9±1.3 and 4.3±1.0 µm for sars-cov-2 and sars-cov, respectively ( fig. 6c ; cc 50 >100 µm, si>20). treatment with peg-ifn-α completely inhibited replication of sars-cov-2, even at the lowest dose of 7.8 ng ml −1 (fig. 6d ). in line with previous results [35, 59] , sars-cov was much less sensitive to peg-ifn-α treatment, yielding only partial inhibition at all concentrations tested (from 7.8 to 1000 ng ml −1 ). overall, we conclude that vero e6 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-2 and sars-cov in vero e6 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 17 years of working with this virus in these cells, sars-cov-2 began to exhibit remarkable phenotypic variation in plaque assays within a few passages after its isolation from clinical samples (fig. 1a) . in addition to the betacov/australia/vic01/2020 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 s5p1 and l8p1, which differed by a single nucleotide substitution in the s protein gene, clearly established that a single s protein mutation (arg682 to gln) was responsible for the observed plaque size difference. this mutation is localized near the so-called furin-like s1/s2 cleavage site (fig. 1b) [61] in the s protein [62] . this sequence constitutes a (potential) processing site that is present in a subset of covs (including sars-cov-2 and mers-cov) but is lacking in others, like sars-cov and certain bat covs [61, 63] . this polybasic motif (prrar↓sv, in sars-cov-2) 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 [64] , which also requires a second cleavage event to occur at the downstream s2' cleavage site [61] . 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 [65] or sars-cov-2 [66] , 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 tmprss2 [67] [68] [69] , thus providing an alternative pathway to trigger viral entry. possibly, this pathway is also employed by our vero e6-cell adapted sars-cov-2 mutants that have lost the furin-like cleavage site, like clone l8p1 and multiple variants encountered in s5p3 (fig. 1a) . these variants contain either single point mutations or deletions of 5 to 10 aa (fig. 1b) , resembling variants recently reported by other laboratories [30, 70, 71] . interestingly similar changes were also observed in some clinical sars-cov-2 isolates that had not been passaged in cell culture [70] . it is currently being investigated why mutations that inactivate the furin-like cleavage site provide such a major selective advantage during sars-cov-2 passaging in vero e6 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/vic01/2020 isolate of sars-cov-2 is the presence of a 10 nt deletion in the 3′ utr of the genome [34] . screening of other available sars-cov-2 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 3′ utr, and in particular to the so-called s2m motif [72] that is conserved among covs and also found in several other virus groups [73, 74] . the s2m 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 s2m) was found to be dispensable for replication in cell culture, but highly relevant for viral pathogenesis in mice [72] . although the impact of this deletion for sars-cov-2 remains to be studied in more detail, these previous data suggest that this mutation need not have a major impact on sars-cov-2 replication in vero e6 cells. this notion is also supported by the fact that the results of our antiviral screening assays (fig. 6) correlate well with similar studies performed with other sars-cov-2 isolates [54, 75, 76] . clearly, this could be different for in vivo studies, for which it would probably be better to rely on sars-cov-2 isolates not carrying this deletion in their 3′ utr. vero e6 cells are commonly used to isolate, propagate and study sars-cov-like viruses as they support viral replication to high titres [77] [78] [79] [80] [81] . this may be due to a high expression level of the ace-2 receptor [82] that is used by both sars-cov-2 and sars-cov [9] and/or the fact that they lack the ability to produce interferon [83, 84] . it will be interesting to evaluate whether there is a similarly strong selection pressure to adapt the s1/s2 region of the s protein when sars-cov-2 is passaged in other cell types. such studies are currently in progress in our laboratory and already established that huh7 cells may be a poor choice, despite the fact that they were used for virus propagation [9, 85] and antiviral screening in other studies [54, 86] . immunolabelling of infected huh7 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-2 stocks propagated in vero e6 cells, at least where it concerns the evolution of the s1/s2 region of the s protein. given the ongoing sars-cov-2 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 1) will be a useful tool during such studies. in general, the subcellular localization of viral nsps and structural proteins (fig. 4 ) and the ultrastructural changes associated with ro formation (fig. 5) were very similar for the two viruses. we also observed comparable replication kinetics for sars-cov-2 and sars-cov in vero e6 cells, although clearly lower final infectivity titres were measured for sars-cov-2 (~50-fold lower; fig. 2 ). nevertheless, rna synthesis could be detected somewhat earlier for sars-cov-2 and the overall amount of viral rna produced exceeded that produced by sars-cov (fig. 3) . this may be indicative of certain assembly or maturation problems or of virus-host interactions that are different in the case of sars-cov-2. 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-2 particles (fig. 5n, r) . our analysis of sars-cov-2 subgenomic mrna synthesis revealed an increased relative abundance of mrnas 7 and 8 (~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 [24] . as in sars-cov, mrna7 of sars-cov-2 encodes for two proteins, the orf7a and orf7b proteins, with the latter presumably being expressed following leaky ribosomal scanning [32] . upon ectopic expression, the orf7a protein has been reported to induce apoptosis via a caspasedependent pathway [87] and/or to be involved in cell-cycle arrest [88] . the orf7b product is a poorly studied integral membrane protein that has (also) been detected in virions [89] . when orf7a/b or orf7a 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 orf7b was reported to provide a replicative advantage in caco-2 and huh7 cells, but not in vero cells [90] . the sars-cov orf8 protein is membrane-associated and able to induce endoplasmic reticulum stress [91, 92] , although it has not been characterized in great detail in the context of viral infection. soon after the emergence of sars-cov in 2003, a conspicuous 29 nt (out-offrame) deletion in orf8 was noticed in late(r) human isolates, but not in early human isolates and sars-like viruses obtained from animal sources [93] [94] [95] . consequently, loss of orf8 function was postulated to reflect an adaptation to the human host. the re-engineering of an intact orf8, using a reverse genetics system for the sars-cov frankfurt-1 isolate, yielded a virus with strikingly enhanced (up to 23-fold) replication properties in multiple systems [96] . clearly, it remains to be established whether the increased synthesis of mrnas 7 and 8 is a general feature of sars-cov-2 isolates, and this indeed also translates into higher expression levels of the accessory proteins encoded by orfs 7a, 7b and 8. 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-19 patients [97] . based on the close ancestral relationship between sars-cov-2 and sars-cov [98] , 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 e6 cells (fig. 6 ) revealed a clear difference, with sars-cov-2 being considerably more sensitive than sars-cov, as also observed by other laboratories [76] . essentially, sars-cov-2 replication could be inhibited by similarly low concentrations of peg-ifn-alpha-2a that inhibit mers-cov replication in cell culture [35] . taken together, our data suggest that sars-cov-2 is less able to counteract a primed type-i ifn response than sars-cov [76, 99] . previously identified inhibitors of cov replication were used to further validate our cell-based assay for sars-cov-2 inhibitor screening. these compounds inhibited replication at similar low-micromolar concentrations and in a similar dose-dependent manner as observed for sars-cov (fig. 6 ). 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 [100] [101] [102] . the clinical efficacy of remdesivir is still being evaluated and, after some first encouraging results [103] , 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-19 patients [104] . both compounds have been used to treat malaria and amebiasis [105] , until drug-resistant plasmodium strains emerged [106] . 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 [68, 107] . an alternative hypothesis to explain their anti-sars-cov activity is based on their impact on glycosylation of the ace2 receptor that is used by sars-cov [56] . finally, as expected, the non-immunosuppressive cyclosporin a analogue alisporivir inhibited sars-cov-2 replication, as demonstrated previously for sars-cov and mers-cov [58] . although the exact mode of action of this inhibitor is unclear, it is thought to modulate cov interactions with members of the cyclophilin family [108] . 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-2 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 2019-ncov and naming it sars-cov-2 bat origin of a new human coronavirus: there and back again coronavirus infection in acute 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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-2 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 1 . the microbiology society is a not-for-profit organization. 2. we offer fast and rigorous peer review -average time to first decision is 4-6 weeks. 3. our journals have a global readership with subscriptions held in research institutions around the world. 4. 80% of our authors rate our submission process as 'excellent' or 'very good'. 5. 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-275863-qos9vu3r authors: dejnirattisai, wanwisa; webb, andrew i.; chan, vera; jumnainsong, amonrat; davidson, andrew; mongkolsapaya, juthathip; screaton, gavin title: lectin switching during dengue virus infection date: 2011-06-15 journal: j infect dis doi: 10.1093/infdis/jir173 sha: doc_id: 275863 cord_uid: qos9vu3r dengue virus receptors are relatively poorly characterized, but there has been recent interest in 2 c-type lectin molecules, dendritic cell–specific intercellular adhesion molecule 3 (icam-3)–grabbing nonintegrin (dc-sign) and its close homologue liver/lymph node–specific icam-3–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 .100 countries, with some 2.5 billion people at risk of infection. dengue is an arthropod-borne flavivirus that can be subdivided into the 4 major serotypes (den-1-den-4). 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 [1] [2] [3] [4] . levels of a number of cytokines, such as interferon c and tumor necrosis factor a [5, 6] , 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 [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] . dc-sign polymorphism has been shown to be associated with the disease severity [19] . clec5a has also recently been described as a proinflammatory receptor for denv that contributes to lethal disease in a mouse model [20] . as dengue virus circulates between 2 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 c6/36 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-2 strain, 16681, was grown in c6/36 cells, vero cells, and monocyte-derived dendritic cells (dcs). cell-free supernatants were used either neat or after concentration by ultracentrifugation at 45,000 rpm for 4 h at 4°c, and the virus pellet was resuspended in 1.5% fetal bovine serum (fbs)/ leibovitz l-15. to concentrate large volumes of low-titer denv supernatant, denv were precipitated with 10% polyethylene glycol 8000 (sigma) before ultracentrifugation. u937 were maintained in 10% fbs/roswell park memorial institute medium (rpmi). nih/3t3, and dc-sign and l-sign nih/3t3 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 10% fbs/ dulbecco's modified eagle's medium. all media were supplemented with 2 mmol/l l-glutamine, 100 u/ml penicillin, and 100 u/ml streptomycin. the viral titers were determined by a focus-forming assay. briefly, virus was serially diluted and incubated with vero cells for 2 hours at 37°c. the monolayers were then overlaid with 1.5% carboxymethylcellulose and incubated at 37°c for 2 days. virus foci were stained with anti-e antibody followed by peroxidase-conjugated anti-mouse immunoglobulin (ig) and visualized by the addition of 3,3#-diaminobenzidine tetrahydrochloride substrate. dcs were cultured as described elsewhere [21] . human cd14 1 monocytes were cultured in rpmi 1640 with 20 ng/ml rhugm-csf (first link) and 25 ng/ml rhuil-4 (ebioscience). the appropriate phenotype of immature dc (ie, lacking cd14 1 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 1 for 24 h. cells were washed twice in facs wash (fw; phosphate-buffered saline [pbs] containing .5% bovine serum albumin, 2% fbs, 1% human serum, .01% nan 3 ). cells were fixed with 4% paraformaldehyde/pbs for 10 minutes and permeabilized with .5% saponin in fw for 10 minutes. cells were then incubated with anti-ns1 mab or with anti-e mab followed by anti-mouse igg/pe (dakocytomation) in .5% saponin/fw. cells were resuspended in fw and analyzed by facscan (becton dickinson). data were analyzed by using flowjo software (tree star). 3t3 and 293t were incubated with 20 u/ml heparin for 20 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 2 h at room temperature. after washing with ice-cold fw, cells were fixed with 4% paraformaldehyde/pbs, and surface-bound virus was detected with an anti-e mab and anti-mouse igg/pe followed by facs analysis. the pcdna3-ll-sign plasmids expressing 7 (n7) and 5 (n5) tandem neck region repeats or a mixture of n7 and n5 plasmids (ratio 1:1) were transfected into 293t cells using the fugene6 (roche). l-sign expression was verified with l-sign-specific antibody (clone 120604, r&d systems). denv supernatant was precleared with protein a-agarose for one h at 4°c. bead-free supernatant was incubated with 10 lg of 4g2 at 4°c for 2 h followed by protein a-agarose for 1 h. the beads were washed with .05% tween/pbs 3 times and eluted with nonreducing loading buffer. the sample was run on nonreducing 10% 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 [22] . digested proteins were separated by 10% 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 2 distinct stages of pathogenesis, we compared viruses from c6/36 (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, 293t, or dcs at an moi of 1. the percentage of infected cells was monitored by cytofluorometry staining of the intracellular nonstructural dengue antigen ns1, which is produced only following productive infection. insect-derived virus was equally competent at infecting the 3 cell types with high efficiency ( figure 1a-c) . surprisingly, dc-produced virus was not able to reinfect dcs but was nevertheless fully competent at infecting 293t and vero cells. the lack of infectivity of dc-produced virus on dcs was also shown using a different mab, 4g2, which reacts with an epitope on dengue envelope protein ( figure 1c , lower panel). a time course of infection was performed where dcs or vero cells were infected with virus produced in c6/36, 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 1d -e). at 24, 48, and 72 h the dc-produced virus showed a much reduced ability to infect dcs when compared with virus produced in c6/36 or vero cells. the experiments we have described above used the dengue serotype 2 strain 16681. to see whether these results could be generalized, we checked infection of both dcs and vero cells with dengue serotype 2 strain new guinea c, serotype 1 strain hawaii, serotype 3 strain h87, and serotype 4 strain h241. infection efficiency was measured by facs at 24 and 48 hours following infection with virus produced in either c6/36 cells or dcs (figure 2a-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 c6/36-produced viruses were used to infect dcs. in these experiments using mixed viruses the dcs were again susceptible to infection, presumably by the c6/36produced virus fraction ( figure 2c ). 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 4g2. virus produced in c6/36 cells could bind to dcs, while binding of dc virus back onto dcs was almost completely absent ( figure 2d ). 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 3t3 cells expressing dc-sign and included in these assays the related c-type lectin l-sign ( figure 3a ), 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 3t3 cells ( figure 3b ). similar to the lack of observable infection of dcs, the dc-derived virus showed a much lower level of infection on dc-sign-expressing 3t3 cells, with ,2% infection. surprisingly, however, this virus progeny was still able to infect cells expressing l-sign, with up to 13% 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 3t3 cells via either dc-sign or l-sign ( figure 3c ). finally, we tested the binding of insect-and dc-produced virus to the 3t3 transfectants ( figure 3d ). 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 c6/36, vero, and dc, purified virus was tested against a panel of lectins with differing carbohydrate-binding specificities by western blot ( figure 4a ). among the 5 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 a2,6 linkage and a2,3 linkage, respectively, which may occur on complex-type n-glycans. dsa recognizes repeating n-acetyllactosamine (galactosyl b1,4 n-acetylglucosamine) sequences; these may also occur on complex-type n-glycans. pna, unlike dsa, binds preferentially to b1,3-linked terminal galactose, such as galactosyl b1,3 n-acetylgalactosamine, a sequence commonly occurring on o-glycosylated proteins and gangliosides. virus produced in c6/36 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 3 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 a2,6-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 60 kda; we did not see any signal at 19 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 3 or more terminal mannose residues, or pngase f, which removes all simple or complex n-linked glycan ( figure 4b ). dc-produced virus showed a mobility shift of around 3 or 6 kda corresponding to the addition of 1 or 2 complex carbohydrates at positions 67 and 153. 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 2 or 4 kda higher than the pngase f-digested material indicated, as with dc-produced virus, that envelope had either 1 or 2 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 .3 terminal mannose residues, whereas others were endo h resistant and contained more heavily processed structures, which is in agreement with a recent report [23] . we conclude from these experiments that in c6/36, vero cells, and dcs both n-linked glycosylation sites can be used but that a single site is used in 50% of cases. envelope from dc-produced virus contains complex, highly processed endo h-resistant carbohydrate. however, in both c6/36 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 3 and 9 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 [24] . 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 2 different-length l-sign alleles simultaneously. we examined this by transient transfection, and 293t cells were used for these assays as they can be transiently transfected to a high level. 293t were transfected with l-sign containing 5 or 7 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 5 ). 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. c6/36-and dc-derived viruses were incubated with increasing levels of pooled convalescent dengue immune serum and subsequently used to infect u937, 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 6a ). in a final series of experiments we investigated whether dc-produced virus could be induced to infect primary human dcs by ade ( figure 6b ). dc-produced virus could be enhanced to infection by .20-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 [25] . 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 [26] . l-sign, like dc-sign, binds to intercellular adhesion molecule 3(icam-3) and is thought to establish cellular interactions with icam-3-expressing t cells. l-sign is able to capture a variety of viruses ( [27] [28] [29] [30] [31] . 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 [32] . the dengue e protein has 2 potential n-linked glycosylation sites at positions 67 and 153. cryo-electron microscopy reconstructions of dengue virus complexed to soluble dc-sign show interaction with the glycan at position 67 [33] . 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 [23, [34] [35] [36] . we show that in c6/36, vero cells, and dcs either one or both sites are used for the dengue 2 serotype 16681. although both glycosylation sites can play important roles in infectivity and viral replication, functional studies have confirmed the importance of asn-67 for infection of dc-sign-expressing cells [33] . wnv e protein contains a single n-linked glycosylation site at position 154 that is absent from some virus strains [37] . 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 154 this also showed differential specificity for l-sign when expressed in mammalian cell lines, perhaps mediated by the presence of n-acetylglucosamine-terminated structures [29] . 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 [38] . 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 [39] . 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 [29] . in humans, l-sign expression is restricted to endothelial cells beneath the subcapsular sinus in lymph nodes [40] , sinusoidal endothelial cells in the liver [27, 41] , alveolar and endothelial cells in the lung [42] , and capillaries in the villous lamina propria of terminal ileum and peyer patches [43] . 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 [44, 45] . 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 cd4 1 t cells [28, 41] . 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 ns3 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-1beta and ifn-gamma as predictive factors for severity high levels of stnfr p75 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 (cd209) mediates dengue virus infection of human dendritic cells the mannose receptor mediates dengue virus infection of macrophages dengue 1 virus binding to human hepatoma hepg2 and simian vero cell surfaces differs dengue virus entry into liver (hepg2) cells is independent of hsp90 and hsp70 heat shock protein 90 and heat shock protein 70 are components of dengue virus receptor complex in human cells identification of grp 78 (bip) as a liver cell expressed receptor element for dengue virus serotype 2 bacterial lipopolysaccharide inhibits dengue virus infection of primary human monocytes/macrophages by blockade of virus entry via a cd14-dependent mechanism dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (dc-sign)-mediated enhancement of dengue virus infection is independent of dc-sign internalization signals dendritic-cell-specific icam3-grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses a variant in the cd209 promoter is associated with severity of dengue disease clec5a is critical for denguevirus-induced lethal disease a complex interplay among virus, dendritic cells, t cells, and cytokines in dengue virus infections histidine 39 in the dengue virus type 2 m protein has an important role in virus assembly n-linked glycans on dengue viruses grown in mammalian and insect cells homozygous l-sign (clec4m) 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 3-grabbing nonintegrin (dc-sign)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes hiv-1 infection l-sign (cd209l) and dc-sign (cd209) mediate transinfection of liver cells by hepatitis c virus the location of asparagine-linked glycans on west nile virions controls their interactions with cd209 (dendritic cell-specific icam-3 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 1 and dengue 2 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-67 during viral propagation envelope protein glycosylation status influences mouse neuroinvasion phenotype of genetic lineage 1 west nile virus strains enhanced immune recognition of cryptic glycan markers in human tumors the b7 homolog butyrophilin btn2a1 is a novel ligand for dc-sign dynamic populations of dendritic cell-specific icam-3 grabbing nonintegrin-positive immature dendritic cells and liver/lymph node-specific icam-3 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 cd209l (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-331680-qlzhtxs0 authors: goryachev, a.n.; kalantarov, s.a.; severova, a.g.; goryacheva, a.s. title: potential opportunity of antisense therapy of covid-19 on an in vitro model date: 2020-11-03 journal: biorxiv doi: 10.1101/2020.11.02.363598 sha: doc_id: 331680 cord_uid: qlzhtxs0 data on potential effectiveness and prospects of treatment of new coronavirus infection of covid-19 caused by virus sars-cov-2 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 50 100 nucleotides from the 5’-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-2 has been identified, the suppression of which with the help of an antisense oligonucleotide can be effective in the treatment of covid-19. the study of the synthesized antisense oligonucleotide 5’ 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 2 mg / ml. at a dosage of 1 mg / ml, viral replication is reduced by 5-13 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 [12] . 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 100 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 [6, 8] . 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-2. considering that sars-cov-2 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 [1, 9] . prerequisites for the use of antisense therapy in the treatment of covid 19 exist. coronavirus infections have previously caused outbreaks of epidemics. in particular, the outbreak of sars (severe acute respiratory syndrome) in china in 2002-2003 was caused by coronavirus sars-cov, the outbreak of middle eastern respiratory syndrome (mers) was also caused by coronavirus [3, 14] . 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 [10, 11, 13] . in work (10), 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-tor2 (genbank ay274119). the nucleotide sequence of the antisense preparations examined is shown in table 1 : table 1 antisense drugs investigated in work (10) name of the antisense drugs nucleotide sequence (5` -3`) №№ numbers of blocked viral rna sites of sars-cov -tor2 trs1 gttcg tttag agaac agatc 56-76 trs2 taaag ttcgt ttaga gaacag 53-72 these sequences are complementary to the following viral nucleic acid sequences (table 2) : table 2 genetic target sequences of the coronavirus genome for trs1 and trs2 drugs name nucleotide sequence of virus rna (5 '-3') site trs1 gatctgttctctaaacgaac site trs2 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 2) to the sequence of a genome of the sars-cov-2 virus (covid-19) the existence of these sequences almost in all strains of a virus allocated at patients during a pandemic of 2019-2020 was revealed (appendix 1, the nucleotide sequences are highlighted in red color and a frame). blast analysis (https://blast.ncbi.nlm.nih.gov/blast.cgi ) showed 100% 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 trs1 and trs2, which were studied in 2005 by the collective [10]. however, starting from mid-march 2020, changes have been observed in the virus strains associated with the loss of sites from the 5` end. in particular, the mt263462 usa: wa 2020-03-23 virus sample did not have the regions designated as trs1 and trs2. 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-2 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. 1. selection of nucleotide sequence. the nucleotide sequence that was intended to block in the sars-cov-2 virus was selected next to the trs1 and trs2 genes at a distance of four nucleotides from the final region (3 '-end) of the trs2 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 trs1 and trs2 sites in work [10] . blast analysis on the genbank (ncbi) showed presence of this sequence at genomes of all sequenced samples sars-cov-2. this sequence has the form: 5`-ctg tgt ggc tgt cac tcg gct in the investigated nucleotide sequences of coronaviruses in appendix 1, this sequence is highlighted in green. the complementary sequence of the antisense oligonucleotide preparation for treatment has the form: 5`-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 [5] , it was found that over a period of 210 nucleotides from the 5 '-end of the viral rna sequence, nucleotides can theoretically form 38 -39 hairpins, from which the sequence we study can be involved in 6 hairpins, while the sequences trs1 and trs2 can be fragments of 16 and 11 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 (10). 2. synthesis of the drug. the synthesis of an antisense oligonucleotide with phosphorothioate protection of the side sugar-phosphate chain 5'-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. 1312 from 04/14/2020). the total amount of 21-membered oligonucleotide synthesized was 15.371 mg (2.38 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. 0044/20 of 09/11/2020). 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-2 rna by real-time pcr. in the experimental work, a transplantable cell line of the kidney of the african green monkey (chlorocebus aethiops) vero-e6 was used. cell cultivation was carried out in a cell growth medium supplemented with fetal bovine serum (fbs) (final concentration 10%). the studies used a pandemic strain of human coronavirus sars-cov-2 "gk2020/1" passage 4, with an infectious activity of 10 6 tcid 50 /ml (tissue cytopathogenic doses) for vero e6 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 e6 cell culture for 96 hours at 37° c in a 5% co 2 atmosphere. infectious activity was determined according to the methods recommended by who. the cytotoxic action of the preparation in vero е6 cell culture was determined using 96-well culture flat-bottomed plates in which vero е6 cells were placed at 12,000 cells/well in a volume of 100 μl of freshly prepared complete medium. cultivation was carried out by 24 hours at a temperature of 37 °c in the atmosphere of 5% of co 2 . after incubating the cells with the preparations for 96 hours at a temperature of 37 o c in an atmosphere of 5% со 2 , the condition of the cell monolayer was visually evaluated. the culture medium was then removed from the plates and 100 μl of reaction medium and 20 μl of mts (3-(4,5solution were added to each well to the cell culture monolayer. after incubation for 3 hours at 37 o c, the results were taken into account on the biorad automatic reader at a wavelength of 490 nm. reference filter was -630 nm. the concentration of preparations reducing the optical density at 490 nm by 50% compared to the control of cells was taken as 50% of the cytotoxic dose (cc 50 ). 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 50 value). the antiviral activity of the sample was assessed visually under a microscope 96 hours after infection by inhibition of the cytopathic effect of the virus in a vero e6 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 e6 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 1.0 mg/ml; 0.1 mg/ml; 0.01 mg/ml and 0.001 mg/ml, respectively. the vero e6 cells used in the study were grown in 96 well culture plates in a volume of 100 μl full for 24 hours at 37 °c in an atmosphere of 5% co 2 . seed dose -12,000 cells / well. 100.0 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 4 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-2, passage 4, with an infectious activity of 10 6 tcid 50 / ml for vero e6 cells to the plates with a monolayer of vero e6 cell culture: 10 1 -10 6 the suspension was diluted by sequential transfer in test tubes with the required amount of the reaction medium -900 µl of the reaction medium and 100 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 e6 according to pcr rna sars-cov-2, 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-2 rna by pcr was carried out by taking 200 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-2 rna in the culture liquid when exposed to the drug: the presence of sars-cov-2 rna (ct value more than 0), the absence of sars-cov-2 rna (ct value is absent). evaluation of the cytotoxicity of the drug at various concentrations was determined by incubating the drug with vero e6 cells for 96 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 e6 cells, an analytical curve was constructed, from which the cc50 was determined. determination of the cytotoxicity of the antisense oligonucleotide substance by visual assessment of the state of the monolayer of the vero e6 cell culture under an inverted microscope did not reveal significant changes in the morphology of cells at a substance concentration of 2 mg/ml and below after 96 hours of incubation of the preparation with cells (table 3) . determination of the antiviral efficacy of the antisense oligonucleotide according to the treatment scheme (administration of the drug 24 hours after infection) was taken into account by the decrease in the infectious titer of the virus in the culture of vero e6 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-2 virus in the vero e6 cell culture at the tested concentrations. the results of the study of the antiviral activity of the drug by pcr are presented in table. 4. 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 1.0 mg/ml, where the ct value is 13.8 compared to the virus control group (11.4 -10.1). in the course of research, it was found that the antisense oligonucleotide is low toxic to the culture of vero e6 cells. the 50% cytotoxic dose of cc 50 was greater than 2.0 mg/ml (exact cytotoxic dose values not determined). at the same time, the results of visual determination of cytotoxicity of the preparations (cc 50 ) were comparable to the results of determination of сс 50 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 e6 cells. the 50% cytotoxic dose of cc50 was greater than 2.0 mg/ml (exact cytotoxic dose values not determined). at the same time, the results of visual determination of cytotoxicity of the preparations (cc 50 ) were comparable to the results of determination of сс 50 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-2, no statistically significant antiviral effect was found in the therapeutic regimen of the drug addition, because according to (4) the minimum effective virus inhibiting concentration is the concentration of the drug reducing the virus titer by at least 1.5 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 0.001-0.1 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 10.3-11.2 doubling cycles. control ct values in the test with infected cells without the addition of antisense oligonucleotide were 11.4 -10.1 values. at a dosage of the same preparation of 1 mg/ml, the ct value was 13.8. this means that at a dosage of 1 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 2.4-3.7 cycles. this indicates that the dosage of the preparation in 1 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 [7] was a range of 5.3 -13.0 times (2 2.4 -2 3.7 ). that is, at a dosage of 1 mg/ml of the preparation, the viral load of cells can be reduced by 5.3-13 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-19 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 1% [12]. the estimated human dosage is calculated based on the following considerations: the sars-cov-2 (covid-19) 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 100 thousand viral particles can multiply, each of which can infect another cell [2]. the total area of the lungs and respiratory tract is, on average, 100 m 2 (10 8 mm 2 ). the surface density of alveolocytes per 1 mm 2 is approximately 10 thousand cells (10 4 ). thus, the total number of cells in the lungs and respiratory tract is 10 12 cells. if we assume that all cells are infected (100,000 viral particles) and 1 drug molecule is needed for each viral rna, the total number of drug molecules per dose per adult is 10 17 , or 16 nmol (102 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 [12] . 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. 1. 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. 2. oligonucleotides previously studied for sars (10) are complementary to the nucleotide sequences of the viral rna of the new coronavirus infection covid-19 in almost all samples and, theoretically, can be considered as drugs for the treatment of the new coronavirus infection covid-19. investigation of an antisense oligonucleotide with phosphorothioate protection 5`-agccgagtgacagccacacag, complementary to the region of viral rna near the 5`-end, showed extremely low toxicity. the cc 50 dose in studies on vero e6 cell culture was more than 2 mg/ml. 4. the study of the antiviral activity of antisense oligonucleotide on vero cell culture e6 according to real-time pcr showed that at a dosage of 1 mg/ml, the reduction in viral sars-cov-2 endothelial infection causes covid-19 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 rp30 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-2 genome and drive rna degradation antisense morpholino-oligomers directed against the 5` 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-343515-fad1yyqx authors: felgenhauer, ulrike; schoen, andreas; gad, hans henrik; hartmann, rune; schaubmar, andreas r.; failing, klaus; drosten, christian; weber, friedemann title: inhibition of sars–cov-2 by type i and type iii interferons date: 2020-10-09 journal: j biol chem doi: 10.1074/jbc.ac120.013788 sha: doc_id: 343515 cord_uid: fad1yyqx the recently emerged severe acute respiratory syndrome coronavirus-2 (sars–cov-2) is the causative agent of the devastating covid-19 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-2 and compared them with those against sars–cov-1, which emerged in 2003. using two mammalian epithelial cell lines (human calu-3 and simian vero e6), we found that both ifns dose-dependently inhibit sars–cov-2. in contrast, sars–cov-1 was restricted only by ifn-α in these cell lines. sars–cov-2 generally exhibited a broader ifn sensitivity than sars–cov-1. moreover, ruxolitinib, an inhibitor of ifn-triggered janus kinase/signal transducer and activator of transcription signaling, boosted sars–cov-2 replication in the ifn-competent calu-3 cells. we conclude that sars–cov-2 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-19. we tested the effect of type i ifn against sars-cov-2 compared with the sars-cov-1 from 2003. two different cell lines were employed, namely the human bronchial epithelial calu-3 and the primate kidney epithelial vero e6. the cells were first treated for 16 h with 100, 500, or 1000 units/ml of recombinant human ifn-a(b/d) and then infected with the viruses at a multiplicity of infection (moi) of 0.01 plaque forming units (pfu)/cell to obtain multistep growth. virus titers in supernatants were determined 24 h later, when titers are reaching a plateau (see below). the data of three biological replicates are shown in fig. 1 . 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-2 (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-1 (light gray bars), titers were also affected. however, at least in vero e6 cells, the reduction of sars-cov-1 appears to be weaker than the reduction of sars-cov-2 (fig. 1b) . observations were similar when the input moi was reduced to 0.001 (fig. s1 ), except that titers of sars-cov-1 in calu-3 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-2 than against sars-cov-1. to further investigate the potential differences between the viruses, we repeated the experiment three times more with the intermediate dose of 100 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. 2, a and b) showed again that (i) both viruses are reduced by ifn (comparison of 0 versus 100 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-2 is more ifn-sensitive than sars-cov-1. the predominant antiviral cytokine (4, 5) . 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 (5, 17) . ifn-l was previously shown to have activity against coronaviruses (11, 18, 19) and proposed as potential covid-19 treatment (20) . hence, we compared the sensitivity of the two sars-coronaviruses also to recombinant human ifn-l. as shown in fig. 3a , pretreatment with 10 or 100 ng/ml ifn-l exhibited only in vero e6 cells a dose-dependent inhibitory effect on sars-cov-2. for sars-cov-1, 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 10 ng/ml and analyzed the data after pooling with the previous 10 ng/ml ifnl experiment (fig. 3b ). 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-2 and the lack of an effect for sars-cov-1. our data thus show that ifn-l can inhibit sars-cov-2 but not sars-cov-1. a recent study on the host cell interactome of sars-cov-2 identified a number of human proteins for which food and drug administration-approved drugs are available (21) . ruxolitinib, a compound known to target the type i and type iii ifntriggered jak/stat signaling pathway (22) , was among the proposed inhibitors of virus-host cell interactions (21) . because virus inhibition by an ifn inhibitor seems counterintuitive, we aimed to clarify the influence of this compound on sars-cov-2 replication. cells were pretreated with 1 mm ruxolitinib for 16 h and infected at the two different mois, and titers were measured 24 or 48 h later. as shown in fig. 4 , with this setting titers in nontreated controls are already reaching a plateau at the 24-h time point. in calu-3 cells, ruxolitinib had a (15) . our data thus indicate that (i) if anything, ruxolitinib is an enhancer rather than an inhibitor of sars-cov-2 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 e6 cells. our observations so far suggest that sars-cov-2 is consistently more sensitive to ifns than sars-cov-1. 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. 5) shows that calu-3 cells have a very similar reaction to both types of ifn concerning phosphorylation of stat1 and stat2 and expression of the ifn-stimulated mxa and isg15. vero e6 cells also responded to ifn-l as expected (23) , but the isg response was lower than to ifn-a. moreover, in nontreated calu-3 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 e6 compared with the calu-3 (fig. 5) . 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 (5, 17) . the recently emerged sars-cov-2 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-2 replication, with effects that in our hands were consistently more profound than against the sars-cov-1 from 2003. 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-2 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 (24) . 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 3 . sensitivity of sars-cov-2 and sars-cov-1 to type iii ifn. a, experiments were performed as described for fig. 1 , except that recombinant human ifn-l was used. log-transformed titers of each virus dose-response experiment with concentrations of 10 and 100 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 10 ng/ml ifn-l were performed, and the resulting titer data were pooled with the 10 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 (25) . 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 (5, 17) . 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-2 replication in cell culture (26) (27) (28) . also in earlier in vivo studies with sars-cov-1, both type i and type iii ifns were shown to be important for the control of infection or the associated disease (29-33). clinical data on the usage of type i ifn against sars-cov-1 or the related mers-cov, however, are limited, are not always conclusive, or did not show a clear benefit (34) (35) (36) (37) (38) . thus, type iii ifn-l rather than the side effect-prone type i ifns (39) might be considered for clinical testing against sars-cov-2. ruxolitinib was proposed as a potential treatment against sars-cov-2 (21, 40) , and a small clinical trial is underway (clinicaltrials.gov, nct04334044), although case reports were discouraging (41) . the replication boost obtained with ruxolitinib on the ifn-competent calu-3 cells indicates that ruxolitinib is not at all inhibiting sars-cov-2 replication. thus, drugs that interfere with viral host interactors may not necessarily be antiviral but rather boost the infection. calu-3 and vero e6 cells were cultivated in dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum (thermo fisher scientific) in a 5% co 2 atmosphere at 37°c. sars-cov-2 (strain sars-cov-2 /münchen-1.2/2020/984, p.2) (42) and sars-cov-1 (strain sars-fra1, p.2) (43) were grown on vero e6 cells and purified via vivaspin columns (sartorius stedim biotech). the viruses were titrated on vero e6 cells. infection experiments were done under biosafety level 3 conditions with enhanced respiratory personal protection equipment. of note, all cells were tested mycoplasma-negative. the cells were pretreated for 16 h with the indicated amounts of pan-species ifn-a(b/d) (pbl assay science) (44), purified recombinant ifn-l3 (18, 45) , or with 1 mm ruxolitinib (selleckchem). infections were performed at a moi of 0.01 and 0.001. at the indicated times postinfection, cell supernatants were collected and titrated by plaque assay on vero e6 cells. the cells were treated for 24 h with the indicated amounts of ifns or ruxolitinib (added 1 h before ifn) and lysed in t-per protein extraction reagent (thermo fisher) supplemented containing 13 protease inhibitor mixture (c0mplete, roche), 13 phosphatase inhibitor mixture set ii (calbiochem), and sample buffer (35.8 mm tris-hcl, ph 6.8, 7.15% glycerol, 1.43% sds, 1.08 mm bromphenol blue). protein samples were run on 12% acrylamide gels and transferred to polyvinylidene fluoride membranes (millipore) via semidry blotting. after blocking in tbs with 5% bsa (for detection of phospho-stats, mxa, and total stat2) or milk powder (all other detections), primary antibody staining was performed overnight at 4°c. the membranes were washed in tbs with 0.1% tween 20, stained with secondary antibodies for 45 min, and washed again in tbs with 0.1% tween 20 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 (46) and statxact ® (version 9.0.0). 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 bmdp3d). for testing the effect of ifn and virus type simultaneously, the two-way anova (program bmdp7d) 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 = 0.05 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 (2020) the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 genome composition and divergence of the novel coronavirus (2019-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-a2b 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 4 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-19 and emerging viral infections: the case for interferon lambda a sars-cov-2 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 1 (interleukin-29) in response to hantaan virus infection peginterferon alfa-2a plus ribavirin for chronic hepatitis c virus infection a randomized phase 2b study of peginterferon lambda-1a for the treatment of chronic hcv infection sars-cov-2 is sensitive to type i interferon pretreatment potent antiviral activities of type i interferons to sars-cov-2 infection 2020) critical role of type iii interferon in controlling sars-cov-2 infection, replication and spread in primary human intestinal epithelial cells. biorxiv crossref 29 sars-cov pathogenesis is regulated by a stat1 dependent but a type i, ii and iii interferon receptor independent mechanism pegylated interferon-alpha protects type 1 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-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study ribavirin and interferon alfa-2a 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-19: combining antiviral and anti-inflammatory treatments side effects of ruxolitinib in patients with sars-cov-2 infection: two case reports transmission of 2019-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 rs30461 single nucleotide polymorphism on ifn-l1 activity and secretion bmdp statistical software manual key: cord-332276-gs80celr 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: 2007-08-20 journal: cell microbiol doi: 10.1111/j.1462-5822.2007.01034.x sha: doc_id: 332276 cord_uid: gs80celr 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, 2004; nelson and white, 2004; festjens et al., 2006; zong and thompson, 2006) . 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., 2003 marra et al., 2003; peiris et al., 2003; rota et al., 2003) . coronaviruses are a family of enveloped, single-, positive-stranded rna viruses with very large genomic size of~30 kb and have been known to infect many animal species as well as humans (siddell, 1995) . one of the most common abnormalities in sars-cov-infected patients is lymphopenia (peiris et al., 2003; chng et al., 2005; chen et al., 2006) , 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., 2004; gu et al., 2005) . 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., 2003; lang et al., 2003; chau et al., 2004; chong et al., 2004; wei et al., 2007) . 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, 2007) . 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., 2004) . 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., 2007) . in various studies, necrosis was also observed in lymphoid tissues and lymph nodes (ding et al., 2003; lang et al., 2003; gu et al., 2005) . 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., 2004; tan et al., 2004; yan et al., 2004; ren et al., 2005; bordi et al., 2006) . in these studies, the vero cell line (or a subclone of vero known as vero e6), 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-2 (ren et al., 2005; bordi et al., 2006) . although necrosis was not observed in sars-cov-infected vero e6 cells (yan et al., 2004) , it has been observed in different tissues obtained from sars-cov-infected patients (ding et al., 2003; lang et al., 2003; chong et al., 2004) . 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) 3b] has been shown to induce necrosis (khan et al., 2006) . apoptosis and necrosis were also observed during the infection of vero cells by the infectious bronchitis virus (ibv), an avian coronavirus (liu et al., 2001) . 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 3b protein of sars-cov, the orf 3b protein of ibv is localized to the nucleus (shen et al., 2003) , although it has not yet been determined whether the latter can induce apoptosis or necrosis. for another human coronavirus, oc43, intracerebral inoculation into mice resulted in acute encephalitis, with neuronal cell death caused by both necrosis and apoptosis (jacomy et al., 2006) . however, infection of mrc-5, diploid human fetal lung cells, seems to induce mainly apoptosis (collins, 2001) . similarly, infection of monocytes/ macrophages in vitro by 229e, yet another human coronavirus, caused mainly apoptosis, although a few necrotic cells were also observed (collins, 2002) . in two independent studies, it was demonstrated that the inhibition of apoptosis, either by caspase inhibitors or by overexpression of the bcl-2 protein, did not affect sars-cov replication in vero cells (ren et al., 2005; bordi et al., 2006) , 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., 2004; kaye et al., 2006) . 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. (2005) reported that the upregulation of pro-apoptotic genes in sars-cov-infected huh7 cells, while the opposite was observed in sars-cov-infected intestinal cell lines, caco-2 and cl-14 (cinatl et al., 2004) . besides these two studies, the transcriptional profiles of apoptosis-related genes in sars-cov-infected vero e6 have also been reported (leong et al., 2005) . 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 2553 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., 2004) . 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., 2005; yu et al., 2005; shao et al., 2006) . one interesting gene that was found to be upregulated is lipocalin 2, 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., 2005) . the authors speculated that the upregulation of lipocalin 2 is a host response to limit tissue damage and inflammation and the overexpression of lipocalin 2 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., 2003; rota et al., 2003) . the first two-thirds of the sars-cov genome encodes the replicase polyproteins (pp1a and pp1ab) that are processed to yield 16 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, 2004) . 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 3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). 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., 2006) . the overexpression of some of the sars-cov proteins could induce apoptosis and/or necrosis, and this is summarized in table 1 . while the 3c-like protease (also known as 3cl pro or m pro ) is the only replicase gene product that has been shown to induce apoptosis (lin et al., 2006) , all the four main structural proteins (s, e, n and m) could induce apoptosis (surjit et al., 2004; chow et al., 2005; yang et al., 2005; zhao et al., 2006) . as shown in table 1 , the experiments were performed in one or two cell lines (table 1) , 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-1 cells (surjit table 1 activates the transcription factor nf-kb (lin et al., 2006) . vero e6 cell line (chow et al., 2005) upregulates the expression of cox-2 (liu et al., 2006a) . jurkat t cell line alters the membrane permeability of mammalian cells (liao et al., 2006) . forms cation-selective ion channels in planar lipid bilayers (wilson et al., 2004) . human pulmonary fibroblast (zhao et al., 2006) not known. nucleocapsid cos-1 cell line (surjit et al., 2004) ; human pulmonary fibroblast (zhao et al., 2006) upregulates the jnk and p38 mapk pathways (surjit et al., 2004) . downregulates erk, phospho-akt and bcl-2 (surjit et al., 2004) . inhibits the activity of cyclin-cyclin-dependent kinase complex and blocks s phase progression (surjit et al., 2006) . activates the transcription factors, nf-kb and ap-1 (he et al., 2003; liao et al., 2005) . upregulates the expression of cox-2 (yan et al. 2006 ). orf 3a vero e6 cell line (law et al., 2005) forms ion channel in xenopus oocytes (lu et al., 2006) . activates the transcription factor nf-kb (kanzawa et al., 2006) . orf 3b cos-7 cell line ; vero e6 cell line (khan et al., 2006) induces cell cycle arrest at the g0/g1 phase . localizes to the mitochondria (yuan et al., 2006a) . orf 7a hela, hepg2, a549, 293t, cos-7 and vero e6 cell lines ; a549 and 293t cell lines (kopecky-bromberg et al., 2006) inhibits cellular protein synthesis (kopecky-bromberg et al., 2006) . induces the phosphorylation and activation of p38 mapk (kopecky-bromberg et al., 2006) . blocks cell cycle progression at g0/g1 phase via the cyclin d3/prb pathway (yuan et al., 2006b) . activates the transcription factor nf-kb (kanzawa et al., 2006) . nf-kb, nuclear factor kappa b; cox-2, cyclooxygenase-2; jnk, c-jun n-terminal kinase; p38 mapk, p38 mitogen-activated protein kinase; erk, extracellular-signal-regulated kinase; ap-1, activator protein 1. et al., 2004) and human pulmonary fibroblast (zhao et al., 2006) . 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 3a, 3b and 7a, have also been shown to induce apoptosis law et al., 2005; yuan et al., 2005; khan et al., 2006) . again, for 3a and 3b, the studies were performed in only one or two cell lines. the overexpression of the 3a protein in vero e6 (law et al., 2005) and the overexpression of the 3b protein in both cos-7 and vero e6 cells induce apoptosis khan et al., 2006) . the overexpression of orf 3b in vero e6 cells also induces necrosis (khan et al., 2006) . a more extensive range of cell lines, including hela, hepg2, a549, 293t, cos-7 and vero e6, was used to demonstrate that the overexpression of 7a 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 1 ). 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. 1) . for example, the sars-cov e protein has the ability to modulate the membrane permeability of mammalian cells (liao et al., 2006) ion channels in planar lipid bilayers (wilson et al., 2004) . in addition, yang et al. (2005) 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-2 family. as bcl-xl is known to be a critical inhibitor of mitochondrial damage following apoptotic stimuli (dejean et al., 2006) , 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-229e (hcov-229e), can also form ion channels in lipid bilayers (wilson et al., 2006) . interestingly, the mhv e protein has also been shown to induce apoptosis and alter membrane permeability (madan et al., 2005) . the overexpression of the sars-cov n protein could upregulate the c-jun n-terminal kinase (jnk) and mitogen-activated protein kinase (p38 mapk) pathways and downregulate the expression levels of extracellularsignal-regulated kinase (erk), phospho-akt and bcl-2 (surjit et al., 2004) . further investigations revealed that it could inhibit the activity of cyclin-cyclin-dependent kinase complex and block s-phase progression (surjit et al., 2006) . the n protein could also activate the transcription factors, nuclear factor kappa b (nf-kb) and activator protein 1 (ap-1) (he et al., 2003; liao et al., 2005) . as these transcription factors regulate a wide variety of cellular processes, including cell proliferation, differentiation and apoptosis (hess et al., 2004; perkins and gilmore, 2006; tergaonkar, 2006) , 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-2 (cox-2), probably through the activation of nf-kb (yan et al., 2006) . the s protein can also upregulate cox-2 expression (liu et al., 2006a) . similar to the e protein, the sars-cov 3a protein has also been shown to form ion channel in xenopus oocytes (lu et al., 2006) . the overexpression of the protein 3b, which was found in both the nucleus and mitochondria, induced cell cycle arrest at the g0/g1 phase . the 7a protein has been shown to block cell cycle progression at g0/g1 phase by reducing the expression of cyclin d3 and phosphorylation of retinoblastoma protein (yuan et al., 2006b) . another study showed that the overexpression of 7a inhibited cellular protein synthesis and induced the phosphorylation and activation of p38 mapk (kopecky-bromberg et al., 2006) . the 3a and 7a proteins could also activate nf-kb and jnk, leading to the enhancement of il-8 and rantes production (kanzawa et al., 2006) . recently, we also demonstrated that the 7a protein interacts with the pro-survival protein, bcl-x l, and the overexpression of bcl-xl prevents 7a-induced apoptosis (tan et al., 2007) . a good correlation between the abilities of 7a deletion mutants to induce apoptosis and to interact with bcl-x l was observed, suggesting that 7a 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 a59 strains and their effects on the central nervous system (see recent reviews by perlman and dandekar, 2005; bergmann et al., 2006) . unlike mhv, infection of young mice (4-8 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., 2004; subbarao et al., 2004; wentworth et al., 2004) . 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-a549 and sars coronaviruses. rat oligodendrocytes, which were obtained from the cg-4 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-2 (bordi et al., 2006) , the mhv-induced apoptosis could be inhibited by the caspase-9 inhibitor and the overexpression of bcl-2 and bcl-x l, indicating that the mitochondrial pathway is involved further downstream (liu and zhang, 2005; liu et al., 2006b) . in 17cl-1, a fibroblast cell line, caspasedependent apoptosis was also observed after infection with both mhv-jhm and mhv-a59 . it was further demonstrated that mhv-a59 infection of 17cl-1 cells activated caspase-8, which in turn cleaved bid, a bh3-domain pro-apoptotic member of the bcl-2 family (chen and makino, 2002) . the resulting tbid p15 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-a59-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 17cl-1 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 7a protein in vero cells resulted in apoptosis, but a mutant virus without the 7a/7b gene still induced extensive cytopathic effects in vero cells, suggesting that 7a does not contribute significantly to viral-induced cell death, at least in this cell culture system (yount et al., 2005) . 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., 1999; barber, 2001; hay and kannourakis, 2002) . 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), oc43, 229e, 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., 1996) . 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 oc43-infected mrc-5 cells or 229e-infected monocytes/macrophages may be partially responsible for the induction of apoptosis (collins, 2001; 2002) . caspase-dependent apoptosis was induced in different cell lines infected with tgev (eleouet et al., 1998; sirinarumitr et al., 1998) . again, many of the apoptotic cells were bystander cells as they were not infected by tgev (eleouet et al., 1998) . 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., 2000) . 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, 2005) . 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., 2005) . however, there is clear evidence for increased production of certain cytokines and chemokines during sars-cov infection (see recent reviews by cameron et al., 2007; chen and subbarao, 2007) , 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, 2007) . 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., 2005) . since the identification of the sars-cov in the year 2003, 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 e6, and was not extensively studied in animal models. similarly, as summarized in table 1 , 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 2557 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., 2003; almazan et al., 2006) 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, 2006) and requirement for infection studies to be performed in biosafety level 3 or 4 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 7a and 7b is not as efficient as wild-type virus in inducing dna fragmentation, implying that 7a and/or 7b 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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(2004) analysis of deaths during the severe acute respiratory syndrome (sars) epidemic in singapore: challenges in determining a sars diagnosis. arch pathol lab med 128: 195-204. chow, k.y., yeung, y.s., hon, c.c., zeng, f., law, k.m., and leung, f.c. (2005) cell death during infection by coronaviruses 2559 key: cord-279975-542qbbgp 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: 2000-03-15 journal: vet microbiol doi: 10.1016/s0378-1135(99)00199-6 sha: doc_id: 279975 cord_uid: 542qbbgp 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. twoand 7-day old, and 2-, 4-, 8and 12-week old specific pathogen-free (spf) pigs were orally inoculated with ped virus isolated from an outbreak. all 2and 7-day old pigs inoculated developed severe watery diarrhea from post-inoculation day (pid) 1 and died between pid 3 and 4. although three of five 2-week old pigs developed diarrhea on pid 1–4, they eventually recovered. in the 4-week old group, three of five pigs had mild diarrhea for 1–2 days. none of the 8and 12-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, 1991) . experimental infections with ped virus isolates have indicated that the virus is a primary etiologic agent of diarrhea in pigs (debouck and pensaert, 1980; debouck et al., 1981; coussement et al., 1982) . the principal features of ped are watery diarrhea, dehydration and high mortality in suckling pigs. in 1978, a coronavirus-like particle was ®rst identi®ed during episodes of epizootic diarrhea in pigs in belgium (pensaert and debouck, 1978) and the uk (wood, 1977; chasey and cartwright, 1978) . ped has subsequently been reported in canada (turgeon et al., 1980) , hungary (horvath and mocsari, 1981) , germany (pospischil et al., 1981) and korea (kweon et al., 1993) . in japan, there was an outbreak of ped-like disease from late 1982 to early 1983 (takahashi et al., 1983; kuwahara et al., 1988) . further outbreaks occurred between late 1993 and 1996 (sueyoshi et al., 1995; tsuda, 1997) . in an acute outbreak of ped in 1996, more than the 39,000 suckling pigs died (tsuda, 1997) . 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 1988 (hofmann and wyler, 1988) . in japan, ped virus was ®rst isolated in vero cell culture in the same manner (kusanagi et al., 1992) . 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 0.3% tryptose phosphate broth, 10% inactivated fetal calf serum (fcs) and antibiotics was used for the propagation of cells. maintenance medium (mm) consisted of eagle's mem with 0.3% tryptose phosphate broth, 0.02% yeast extract, and 10 mg/ml trypsin (difco, usa), unless otherwise stated. established cell lines of vero cells derived from african green monkey kidney (rcv0001) and ma104 from fetal macacus rhesus monkey kidney were used. sb1 and sb2 cells prepared from the bladder epithelial cells of cesarean-derived colostrumdeprived (cdcd) piglets were used for virus isolation at approximately 20±30 passages. sk cells prepared from the cdcd pig kidney were used at approximately 70±80 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 10% pooled homogenates were centrifuged and the resulting supernatants were passed through a 450 nm membrane ®lter. six 1-day old cdcd piglets were orally inoculated with 2 ml of the homogenized samples and sacri®ced on post-inoculation days (pid) 1±3. 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 à808c until experimental inoculation of pigs. the virus stock was approximately 10 5.0 ld 50 /2 ml for a 3-day old pig. for virus isolation, three 7-day old cdcd piglets were inoculated with the virus stock and sacri®ced on pid 1 (second passage in pig). homogenized samples were prepared for virus isolation according to the method described previously (hofmann and wyler, 1988 ). after removal of gm, con¯uent cell cultures in collagen-coated 12-well tissue culture plates (iwaki glass, japan) were washed once with mem. then, the cells were inoculated with 0.2 ml per well of the clari®ed homogenate. after adsorption at 378c for 1 h, the inoculum was removed and the monolayers were washed three times with 2 ml of mem. the cell cultures were fed with 2 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 7 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 2 days old, two at 7 days old, ®ve each at 2, 4, 8 and 12 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 29±308c for 2-and 7-day old pigs and 23±248c 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 2 ml of 100-and 2-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) 4 or 6. vero cell cultures on coverslips were ®xed in acetone for 10 min, about 24 h after virus inoculation. they were then stained with the experimentally inoculated pig serum at 378c for 50 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 378c for 50 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 568c for 30 min before use. serial two-fold dilutions of serum were mixed with an equal volume of z94p5 strain virus suspension containing 200 tcid 50 /0.1 ml. the mixture were incubated at 378c for 1 h and 0.1 ml of virus±serum mixture was inoculated into each of the two wells. following adsorption at 378c for 1 h, the inocula were removed and the monolayers were washed three times with mem. then, 0.1 ml of mm containing 5 mg/ml trypsin was added to each well and the cultures were incubated at 378c for 7 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, 1997) was used as primary antibody. histopathological examination was performed according to routine procedures. in brief, tissue samples were ®xed in 20% neutral phosphate-buffered formalin. thin sections of paraf®n-embedded samples were stained by hematoxylin and eosin. as shown in table 1 , cytopathic agents were isolated in vero, sk and sb1 cell cultures inoculated with second passage intestinal samples. at ®rst passage, cpe was inapparent vero à a sk à à sb1 à sb2 à à à ma104 à à à because of the cytotoxic effect of the inoculum. on passage 2 in vero cell culture, cpe characterized by cell fusion and syncytial formation was detected microscopically after an incubation period of 1±2 days. cpe progressed more slowly in sk and sb1 cell cultures than in vero cell culture. the number of cells which were rounded, detached and fused were increased after 3±4 days of incubation (fig. 1) . although syncytial formation was not apparent under microscope in non-stained cultured cells, fused cells were obvious in sb1 cell culture samples stained with wright giemsa's solution (fig. 1) . vero cells inoculated with viruses isolated in sb1 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. 1 ) 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. 5 in vero cell culture was clone-puri®ed three times by plaque selection and designated z94p5. cpe was not detected microscopically in sb2 and ma104 cells at the ®fth passage. all 2-and 7-day old pigs inoculated with ped virus developed severe watery diarrhea from pid 1 and died between pid 3 and 4 (table 2). ped virus antigen was detected by the ab technique in the small intestine of dead pigs. vomiting was occasionally seen on pid 1±3. non-inoculated control 2-day old pigs kept under the same conditions remained healthy. three of ®ve 2-week old pigs developed diarrhea on pid 1±4 which lasted 2±4 days. although all these pigs were slightly depressed and anorectic after inoculation, they recovered after 1 week. ped virus antigen was detected by the ab technique in the jejunum and ileum of pig no. 9 which was euthanized on pid 5. in the 4-week old group, three of ®ve pigs had mild diarrhea which started on pid 3±5 and continued for 1±2 days. in this group, other clinical signs such as depression and anorexia were not observed. all of 8-and 12-week old pigs showed no clinical signs including diarrhea throughout the experiment. all the pigs in the 2-, 4-, 8-and 12-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 1 or 2 and they peaked between piw 4 and 5 on vn test and on piw 3 on ifa (fig. 2) . 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 sb1 and sk cell cultures. hofmann and wyler (1988) ®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, 1988) . 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 sb1 cells but not in sb2 cells. although ped virus propagated in sb1 cells showed cpe in sb2 cells, the titer in these cells was lower than in sb1 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, 2-and 7-day old pigs inoculated with ped virus developed severe diarrhea and died. the 2-and 4-week old pigs showed mild diarrhea after inoculation and survived. the 8-and 12-week old pigs did not show any clinical signs, but developed antibody against ped virus. coussement et al. (1982) described that 2-or 3-day old cesarian-derived piglets infected oronasally with the cv777 strain of ped virus showed severe diarrhea after an incubation period of 22±36 h. debouck and pensaert (1980) reported that pigs between 1 and 20 days old experimentally inoculated with ped virus developed diarrhea and some pigs younger than 19 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, 1978; takahashi et al., 1983) . in the present study, however, severe diarrhea was only observed in 2-and 7-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, 1997) . in tge virus infection, (shimizu et al., 1978; shimizu and shimizu, 1979) 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, 2±12-week old pigs were kept at 23± 248c 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 cv777 coronavirus enteritis in piglets experimental infection of pigs with a new porcine enteric coronavirus cv777 the pathogenesis of an enteric infection in pigs, experimentally induced by the coronavirus-like agent, cv777 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, cv777, 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-323839-a4oejky0 authors: sasaki, michihito; uemura, kentaro; sato, akihiko; toba, shinsuke; sanaki, takao; maenaka, katsumi; hall, william w.; orba, yasuko; sawa, hirofumi title: sars-cov-2 variants with mutations at the s1/s2 cleavage site are generated in vitro during propagation in tmprss2-deficient cells date: 2020-08-28 journal: biorxiv doi: 10.1101/2020.08.28.271163 sha: doc_id: 323839 cord_uid: a4oejky0 the spike (s) protein of severe acute respiratory syndrome-coronavirus-2 (sars-cov-2) 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-2 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, tmprss2. notably, viruses with s gene mutations emerged rapidly and became the dominant sars-cov-2 variants in tmprss2-deficient cells including vero cells. our study demonstrated that the s protein polybasic cleavage motif is a critical factor underlying sars-cov-2 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-2 in tmprss2-expressing cells [6] . in contrast, camostat had no impact on entry of the s gene mutant, del2, into 1 4 0 vero-tmprss2 cells but e-64d treatment resulted in a dose-dependent decrease in del2 1 4 1 entry (fig. 4a) . these results suggested that s gene mutant, del2, can enter vero-tmprss2 1 4 2 cells via cathepsin-dependent endocytosis but not the tmprss2-mediated fusion pathway. parental vero cells that do not express tmprss2 were inoculated with s gene mutant 1 4 4 viruses in the presence of camostat and/or e-64d. addition of e-64d inhibited the entry of 1 4 5 all s gene mutants into both vero-tmprss2 and parent vero cells; by contrast, camostat 1 4 6 had no impact on s gene mutant entry into these target cells (fig. 4b ). these results 1 4 7 8 suggested that, in contrast to wt virus, s gene mutants enter into cells via 1 4 8 cathepsin-dependent endocytosis only, regardless of the presence or absence of tmprss2. because wt virus and s gene mutants showed different sensitivities to the treatment 1 5 0 with camostat, an agent currently under exploration as a candidate antiviral for clinical use 1 5 1 [17], we also examined the impact of other antiviral agents including nafamostat (a 1 5 2 tmprss2 inhibitor) [18, 19] and remdesivir (a nucleotide analog) [20, 21] . antiviral 1 5 3 effects in vero-tmprss2 cells were estimated by a cell viability assay based on the 1 5 4 generation of cytopathic effects [22] . consistent with previous studies [18, 19] , nafamostat 1 5 5 showed higher antiviral efficacy against wt virus than was observed in response to 1 5 6 camostat; however, nafamostat had no antiviral activity against the s gene mutants ( table 1 5 7 1). in contrast, remdesivir inhibited infection of both wt and s gene mutants with similar 1 5 8 ec 50 values (table 1 ). these results indicated that s gene mutants are resistant to the 1 5 9 treatment with tmprrss2 inhibitors, but are sensitive to antivirals that target post entry in an effort to understand the selection mechanisms underlying the generation of these 1 6 4 mutant variants, we estimated the frequency of s gene mutants in virus population of 1 6 5 sars-cov-2 that had undergone serial passage in cultured cells. sars-cov-2 from an 1 6 6 original virus stock was underwent passage once (p1) to four times (p4) in vero or up to 1 6 7 eight times (p8) in vero-tmprss2. nucleotide sequence heterogeneity at the s1/s2 1 6 8 cleavage site was determined by deep sequencing and variant call analysis. more than one 1 6 9 9 million sequence reads from each passaged sample were mapped onto the s1/s2 cleavage 1 7 0 site and analyzed for sequence variation. no sequence variants were observed in virus 1 7 1 populations until p8 in vero-tmprss2 (fig. 5a) . in contrast, nucleotide sequence 1 7 2 deletions around the s1/s2 cleavage site corresponding to del1 and del2 mutants were 1 7 3 observed in all three biological replicates of sars-cov-2 populations passaged in vero 1 7 4 cells (fig. 5a) . notably, wt nucleotide sequences were detected in fewer than 20% of the 1 7 5 isolates evaluated at p2 and the wt was completely replaced with s gene mutants at p4 in 1 7 6 vero cells. these results indicated that sars-cov-2 propagation in vero cells results in a 1 7 7 profound selection favoring the s gene mutants. s gene mutants del3 and r685h were not 1 7 8 identified in the virus populations from p1 to p4. an additional variant del4 with a deletion 1 7 9 of 5 amino acids at a point immediately upstream of the rrar motif (figs. s2a and s2b), 1 8 0 was detected as a minor variant in sample #1 at p2. these results suggest that these specific 1 8 1 mutations occur only at low frequency. we then determined the frequency of s gene mutants in virus populations passaged in 1 8 3 calu-3 and caco-2, which are cells that endogenously express tmprss2 [13, 14] , and also 1 8 4 in 293t-ace2 that do not express tmprss2 [6] . no s gene mutants were identified in 1 8 5 sars-cov-2 passaged in calu-3 and caco-2 until p4; by contrast, s gene mutants 1 8 6 emerged at p2 in 293t-ace2 cells (fig. 5b) . we also identified an additional variant 1 8 7 r682p carrying a single amino acid substitution at the rrar motif (figs. s2a and s2b) at 1 8 8 p3 and p4 in 293t-ace2 cells (fig. 5b) . taken together, these results suggest a strong 1 8 9 association between tmprss2 deficiency and the emergence of s gene mutants. trypsin is a serine protease that is typically added to culture medium to induce 1 9 1 cleavage and activation of viral proteins, including the hemagglutinin (ha) protein of 1 9 2 influenza virus and the fusion (f) protein of paramyxovirus to promote growth in 1 9 3 tmprss2-deficient cells [23] . recent studies report that trypsin treatment activates 1 9 4 sars-cov-2 s protein and induces syncytia formation in cells that transiently express the 1 9 5 virus s protein [7, 24] . as such, we examined whether exogenously added trypsin could 1 9 6 compensate for tmprss2 deficiency and thus inhibit the emergence of s gene mutants 1 9 7 during sars-cov-2 propagation in vero cells. deep sequencing analysis revealed that s 1 9 8 gene mutants did emerge and accounted for the majority of the virus population after p2 in 1 9 9 vero cells cultured in serum-free medium with added trypsin ( in this study, we isolated s gene mutants from sars-cov-2 wk-521, a strain isolated difficult to impossible to maintain sars-cov-2 with the s1/s2 cleavage site in its intact 2 1 6 form. the present study characterized s gene mutants as sars-cov-2 variants that generate 2 1 8 small plaques and that have a narrow range of cell tropism. the phenotypic alterations of s 2 1 9 gene mutants might be explained by noting that the s gene mutants were unable enter target 2 2 0 cells via direct fusion mediated by tmprss2. indeed, a previous study demonstrated that 2 2 1 the polybasic cleavage motif at the s1/s2 cleavage site was indispensable for the entry of 2 2 2 vsv-pseudotyped viruses into calu-3 cells that expressed tmprss2 [7] . further studies 2 2 3 using infectious s gene mutants will provide new insights into the role of the polybasic 2 2 4 amino acid motif at the s1/s2 cleavage site with respect to both sars-cov-2 infection and 2 2 5 its pathogenicity. at this time, many studies are conducted using sars-cov-2 propagated in vero cells. considering the very real possibility that these virus stocks will accumulate s gene 2 2 8 mutations, researchers must pay careful attention to the passage history of any working 2 2 9 stocks of sars-cov-2. moreover, we must be very objective when interpreting the results 2 3 0 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-2 at an moi of 1. after 24 h, cells were fixed with 3.7% buffered formaldehyde, permeabilized with ice-cold 2 8 3 methanol, and incubated with anti-sars-cov-2 s antibody (gtx632604, genetex). cells were inoculated with either wt or s mutants of sars-cov-2 at an moi of 0.1. after 1 h of incubation, cells were washed twice with phosphate-buffered saline (pbs) and 2 9 2 cultured in fresh medium with 2% fbs. the culture supernatants were harvested at 24, 48, 2 9 3 and 72 h after inoculation. virus titers were evaluated by plaque assay. vero-tmprss2 were infected with either wt or s mutants of sars-cov-2 at 4-10 3 1 5 tcid 50 and added to the plates. plates were incubated at for 3 days, and cpe was 3 1 6 determined for calculation of 50% endpoints using mtt assay. the concentration 3 1 7 achieving 50% inhibition of cell viability (effective concentration; ec 50 ) was calculated. the original stock of sars-cov-2 strain wk-521 was serially passaged in vero, vero-tmprss2, calu-3, caco-2, and 293t-ace2 cells in complete culture medium or 3 2 2 (for vero) in serum free dmem supplemented with 0.5 μ g/ml trypsin (gibco); three full-length s protein is cleaved into s1 and s2 proteins at the s1/s2 cleavage site. functional domains (rbd, receptor binding domain; rbm, receptor binding motif) are highlighted. (b) multiple amino acid sequence alignments focused on the s1/s2 cleavage site of wild type (wt) and isolated mutant viruses (del1, del2, del3 and r685h). amino acid substitutions and deletions are shown as gray boxes, and the polybasic cleavage motif (rarr) at the s1/s2 cleavage site is highlighted in red. a red arrowhead indicates s1/s2 cleavage site. (a) vero-tmprss2 cells were infected with sars-cov-2 wt or del2 mutant in the presence of varying concentrations of the tmprss2 inhibitor, camostat, or the cathepsin b/l inhibitor, e-64d, for 1h. at 6 h post-inoculation, the relative levels of viral n protein rna were evaluated quantitatively by qrt-pcr. (b) vero-tmprss2 and vero cells were infected with sars-cov-2 wt or s gene mutants in the presence of 50 μm camostat and/or 25 μm e-64d for 1 h. at 6 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 < 0.05, **p < 0.01, ***p < 0.001. multiple (a) nucleotide and (b) amino acid sequence alignments were constructed based on the sequence of wt and sars-cov-2 variants identified by deep-sequencing (related to fig. 4) . infectious viruses of del4 and r682p were not isolated in this study. nucleotide substitutions and deletions are shown as gray boxes. sequence encoding the polybasic cleavage motif (rarr) at the s1/s2 cleavage site is highlighted in red. sars-cov-2 was serially passaged in vero cells in serum free dmem containing trypsin with three biological replicates. nucleotide sequence diversity at viral s1/s2 cleavage site was determined by deepsequencing. sars-cov-2 (r685h) atcagactcagactaattctcctcggcgggcacatagtgtagctagtcaatccatcattgc multiple nucleotide sequence alignment of s1/s2 cleavage site of wild type and isolated sars-cov-2 mutants nucleotide substitutions and deletions are shown as gray boxes. sequence encoding the polybasic cleavage motif (rarr) at the s1/s2 cleavage site is highlighted in red sars-cov-2 (r682p) atcagactcagactaattctcctccgcgggcacgtagtgtagctagtcaatccatcattgc sars-cov-2 (r682p) ecdipigagicasyqtqtnspprarsvasqsiiaytmslgaensvaysnns ecdipigagicasyqtqt----------sqsiiaytmslgaensvaysnns sars-cov-2 (del2) ecdipigagicasyqtqtnspr-------qsiiaytmslgaensvaysnns sars-cov-2 (del3) ecdipigagicasyqtqtnsprrarsva---iiaytmslgaensvaysnns sars-cov-2 (r685h) ecdipigagicasyqtqtnsprrahsvasqsiiaytmslgaensvaysnns key: cord-274110-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: 2010-04-26 journal: vaccine doi: 10.1016/j.vaccine.2010.02.092 sha: doc_id: 274110 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 [1] ). in the u.s. wnv activity in human, bird, companion animals or mosquito has been reported since 1999 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) [2] . the flavivirus genome is a positive-polarity, single-stranded rna molecule of about 11,000 nucleotides (nt), which functions as mrna for translation of the viral proteins. genomic rna is infectious when introduced into susceptible cells by transfection [3] . for replication and pathogenesis studies, reverse genetic systems have been established for several members of the genus [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . 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 [4] , den-1 [17] , den-2 [6, 8, 10] , den-4 [11] , tbev [13, 15] , kun [9] , mve [7] and wnv lineage i [19] and ii [21] , cdna comprising the full genome was stably cloned into bacterial expression plasmids, whereas in other reports [5, 8, 13, 18, 20] 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 [22] . 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 [6, 7, 9, 13, 14, 16, 19] . in 2002, 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 7.5 kb genome was assembled from overlapping oligonucleotides and yielded infectious virus after transcription of genomic rna and inoculation into cell lysates [23] . taking advantage of the rapid progression of gene synthesis technology (for review [24] ), 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 11,029-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 ny99-flamingo 382-99 was obtained from centers for disease control (cdc, atlanta) corresponding to genbank accession #af196835. 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-81), bhk (atcc ccl-10) and c6-36 (eceacc 123.p. #03d016) 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 f12 medium. six dna fragments corresponding to wnv strain ny99-flamingo 382-99 (genbank accession #af196835) were generated by chemical synthesis (geneart, regensburg, germany). plasmid p5 tl-ab carried dna corresponding to wnv genomic sequence nt 1-1792, plasmid p5 tl-cd to nt 1789-3632, plasmid p3 tl-ab to nt 3622-5801, plasmid p3 tl-cd to nt 5792-8028, plasmid p3 tl-ef to nt 8022-10,025 and plasmid p3 tl-gh to nt 10,022-11,029. three silent mutations at positions 8859, 8862, and 8880 were introduced to knock out an ecori site and to create an styi site as described previously [19] . for assembly of plasmid pwnvsyn-5 tl (containing the 5 third of the wnv coding sequence) an asci and bsai cut fragment of p5 tl-ab was ligated to a bbsi and paci cut fragment of p5 tl-cd (fig. 1b) . 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 pbr322-pl (derived from plasmid pbr322 engineered to contain a matching polylinker between the unique restriction sites ecor1 and eagi resulting in the partial deletion of the tet r gene) yielding pwnvsyn-5 tl. this plasmid contains the 3 two-thirds of the wnv coding sequence and was generated in three steps: (i) an asci and bsai cut fragment of p3 tl-ab was ligated to a bsai and paci cut fragment of p3 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 p3 tl-ef was ligated to a paci and btgzi cut fragment of p3 tl-gh. the resulting 3 tl-efgh ligation product was amplified by pcr using 5 and 3 flanking primers, digested with spei and xbai and integrated in pbr322-pl, leading to plasmid pbr322-3 tl efgh (fig. 1b) . (iii) the final pwnvsyn-3 tl was generated by introduction of the 3 tl-abcd fragment (derived from ppcrscript3 tl-abcd) into pbr322-3 tl-efgh, taking advantage of the unique restriction enzymes spei and bamhi (fig. 1c) . all plasmids were amplified in bacterial strain hb101 (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 1.8 kv, 25 f and 200 . sequence analysis was carried out using a 3130xl genetic analyzer (abi) using bigdye terminator v3.1 cycle sequencing kit (abi). pwnvsyn-3 tl was linearized with xbai followed by mung bean nuclease digestion to remove single stranded nucleotide overhangs in order to generate the correct 3 end of the wnv coding sequence. the plasmids pwnvsyn-3 tl and pwnvsyn-5 tl were then digested with sphi and the full-length sequence was generated by ligation (t4 ligase; new england biolabs) via the sphi sequence overhangs of the 5 and 3 parts. the ligated dna fragments were extracted with phenol-chloroform twice, precipitated with ethanol and resuspended in nuclease free water. rna was transcribed at 37 • c for 3 h from ligated template dna by t7 polymerase transcription, using t7 megascript kit (ambion). the integrity of rna transcripts was analyzed in 1% agarose gels containing 6% 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 2 × 10 7 cells were resuspended in 800 l of ice-cold pbs, mixed with transcribed rna and transferred to 0.4 gene pulser cuvettes. cells were electroporated with three successive pulses using a genepulser apparatus (bio-rad) with settings of 0.8 kv, 25 f and 200 . to visualize intracellular expression of wnv proteins, cells were infected or transfected. two days later, cells were fixed with acetone-methanol (1:1). cover slips with fixed cells were dried, rehydrated with phosphate-buffered saline and treated with a polyclonal mouse anti-wnv serum (1:50 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 (1:100 dilution; jackson research laboratory). vero or c6/36 cells grown in 175 cm 2 tissue culture flasks were infected with either wnvsyn or wnvwt stock at an moi of 0.0001. the inoculum was removed after 1 h, and 40 ml of fresh medium was added. at various time points (1, 6, 24, 48, 54, 72 and 96 h) 0.5 ml of medium was removed. the infectious virus titer of wnv containing samples was determined by a tcid 50 assay. in brief, serial 10-fold dilutions of virus containing supernatant were inoculated in 96-well microtiter plates seeded with vero cells. after incubation for 7 days at 37 • c and 5% co 2 , 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 50 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 3 × 10 7 tcid 50 by trizol extraction. rna was precipitated with ethanol and the rna pellet was resuspended in 50 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 3 end of the ns5 coding region, the ns2b3 coding region and the 3 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 4) as well as wnvwt preparation (designated cag 6) was prepared in the same manner. ten roller bottles of bhk cells were infected with a moi of 0.0001. for better virus yields ph was adjusted to 7.5 after 1 h of virus adsorption. after 4 days of growth the supernatant was harvested and cleared through a low spin centrifugation step at 2500 rpm. the cleared supernatant was treated with formalin (final concentration 0.005%) for 48 h. next, 30 ml of the inactivated virus was loaded on 5 ml of a 20% sucrose cushion per centrifugation tube (beckman, sw28 tubes). after 2 h centrifugation with 104,000 × 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 50 (ld 50 ) was determined in female 7-weekold balb/c mice. groups of six mice were infected intranasally with 1 × 10 1 , 1 × 10 2 , 1 × 10 3 and 1 × 10 4 tcid 50 of wnvsyn or wnvwt, respectively. survival of mice was recorded for a period of 28 days after infection. the 10-fold virus dilutions were titrated shortly after challenge and were used to calculate the ld 50 values using the computer program graph pad prism 5. protection was determined after immunization of female 7-week-old balb/c mice by subcutaneous injections of formalininactivated wnvsyn or wnvwt vaccines in a volume of 100 l in tbs containing 0.2% al(oh) 3 . mice were challenged intranasally with 10 l of pbs (0.01% human serum albumin) containing 2 × 10 5 tcid 50 wnvwt virus. survival was monitored over a period of 28 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 1:1 with a virus stock suspension adjusted to 1 × 10 2 tcid 50 , incubated for 90 ± 15 min at room temperature and transferred (eight replicates per dilution) to a 96-well microtiter plate seeded with vero cells. the plates were inspected under a light microscope for the presence of cpe after incubation for 6 days at 37 • c and 5% co 2 . the neutralizing titer was calculated by counting cpe negative wells and by usage of the formula nt-titer = (v/2) × 2e((nneg/8) + 0.5) 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 c6/36 cells were seeded in five 175 cm 2 tissue culture flasks and inoculated with individual preparations corresponding to 12 ml of the infectious yield from which the preparations were derived. after a 10 day incubation period at 37 • c and 5% co 2 , supernatant of each flask was titrated by tcid 50 and 2 ml supernatant of each flask was carried onto fresh vero and c6/36 cells. after a 10-day observation period supernatant of each flask was titrated by tcid 50 . 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 50 assays. the amount of wnv antigen in respective samples was determined by means of an elisa double sandwich system. briefly, 96-well microtiter plates were coated by overnight incubation at 2-8 • 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 1 h at 37 • c. after subsequent washing steps a mouse anti-wnv polyclonal serum was applied to the wells and incubated for 1 h at 37 • c. after washing, the wells were incubated with horseradish peroxidase-conjugated donkey anti-mouse igg (jackson immuno research laboratories) for 1 h at 37 • c. after subsequent washing steps, substrate (o-phenylenediamine/h 2 o 2 ) was added, and the enzyme reaction was stopped after 15 min at 37 • c by the addition of 0.25 m h 2 so 4 . the absorbance at 490 nm was measured with an elisa plate reader (bio-tek, winooski, vt, usa) and the antigen content was calculated (kc4 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 3 conditions. experiments were approved by the baxter internal biosafety committee and by the austrian ministry of health (bmfg-76110/0002-iv/b/12/2005). for the construction of a bipartite infectious clone, six contiguous cdna fragments encoding the genome of the lineage i wnv strain ny99 were chemically synthesized and integrated in bacterial expression plasmids (see section 2) according to the cloning strategy outlined in fig. 1 . three silent marker mutations were introduced (see also [19] ) allowing the discrimination of the synthetic virus from the corresponding wild-type isolate (see table 1 ). 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 5 third (nt 1-3632 under control of a t7 promoter) or the 3 two-thirds (nt 3622-11,029) of the wnv genomic sequence, designated as pwnvsyn-5 tl or pwnvsyn-3 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-3 tl and pwnvsyn-5 tl. genome length viral rna was transcribed and the integrity of rna transcripts was analyzed in 1% agarose gels containing 6% formaldehyde. an rna band of approximately 11,000 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 2 days later (fig. 2) . viral protein expression was monitored with a specific polyclonal mouse anti-wnv antibody and a fitc-conjugated second antibody (see section 2). cells infected with moi 0.0001 of wnvwt and were used as staining control. wnvsyn-transfected and wnvwt-infected vero cells exhibited wnv protein expression in approximately 20% of all cells. as expected, viral antigen staining is mainly confined to perinuclear regions of the cells (fig. 2) . immunofluorescence staining is only detectable from replication-and translation-competent viral templates and could not be shown in replication-deficient mutant viral genomes [19, 25] 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 3 days, cell culture medium was harvested and the virus titer was determined on vero cells, yielding a titer of 1.62 × 10 8 tcid 50 /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 0.0001 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. 2d) . in order to analyze the growth properties of wnvsyn and wnvwt, one step growth curves were carried out. susceptible mammalian (vero) and mosquito (c6/36) cells were infected with a moi of 0.0001. viral titers, determined at the time points indicated in fig. 3 , demonstrate that in both cell types the growth kinetics of wnvsyn match exactly those of the wild-type virus. in addition, plaque morphology ( fig. 3a and b) and cpe (not shown) were comparable to the wild-type control. virus grown in vero cells peaked at 48 h posttransfection but declined from day 2 on correlating with the onset of cpe of the infected cells from day 3 on (fig. 3c) . growth kinetics in the mosquito cells was delayed as observed by others [19, 25] , reaching equal titers compared to vero cells at day 4 postinfection (fig. 3d) . 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 7-week-old balb/c mice. for this purpose mice were infected intranasally with virus dilutions corresponding to 2 × 10 5 to 2 × 10 2 tcid 50 per animal. survival was monitored for 21 days postinfection and ld 50 values were calculated. similar mortalities of infected mice induced by the two wnv viruses were observed ( table 2 ). the lethal dose 50 for wnvsyn and wnvwt was 3.6 and table 2 virulence of wnvsyn and wnvwt after intranasal application in balb/c mice. 3.4 log 10 tcid 50 , 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 2). 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. 4b) . the predominant band in these preparations is the envelope antigen (e) migrating in the 60 kda range, the fainter bands representing the pre-membrane (prm) and the dimeric membrane (m) proteins (see also [26] ). 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. 4a and table 3 ). mice were then challenged intranasally with a lethal dose (1 × 10 5 tcid 50 ) 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 63 nanograms of the wnv antigens while 95% of the non-vaccinated controls died. the vaccines clearly induced a dose-dependent protection correlating with nt titers (table 3) . 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. [27, 28] ). 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 [29] , bacteriophage x174 [30] or h1n1 spanish influenza virus [31] , and sars-like coronavirus [32] . on the basis of these studies, we report for the first time the generation of an 11,000 nucleotide long synthetic genome of a member of the family flaviviridae. sequence data from genbank referring to lineage i west nile virus strain ny99 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 [33] ), resulting in a multitude of distinct but related nucleic acids forming a quasispecies [34] . 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 [35] , 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 [36] . wnv -although a biosafety level 3 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 [24] . 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 2 using stably cloned genomic-length cdna characterization of infectious murray valley encephalitis virus derived from a stably cloned genome-length cdna synthesis and characterization of an infectious dengue virus type-2 rna genome (new guinea c strain) completion of kunjin virus rna sequence and recovery of an infectious rna transcribed from stably cloned full-length cdna construction of infectious cdna clones for dengue 2 virus: strain 16681 and its attenuated vaccine derivative, strain pdk-53 infectious rna transcribed from stably cloned full-length cdna of dengue type 4 virus molecular and functional analyses of kunjin virus infectious cdna clones demonstrate the essential roles for ns2a in virus assembly and for a nonconservative residue in ns3 in rna replication infectious cdna clones of tick-borne encephalitis virus european subtype prototypic strain neudoerfl and high virulence strain hypr infectious clone construction of dengue virus type 2, strain jamaican 1409, and characterization of a conditional e6 mutation infectious cdna clone of attenuated langat tick-borne flavivirus (strain e5) and a 3 deletion mutant constructed from it exhibit decreased neuroinvasiveness in immunodeficient mice infectious rna transcripts from fulllength dengue virus type 2 cdna clones made in yeast construction of a full length infectious clone for dengue-1 virus western pacific, 74 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 17d-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: phix174 bacteriophage from synthetic oligonucleotides characterization of the reconstructed 1918 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-273745-mwjh5se7 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: 2014-03-25 journal: virology doi: 10.1016/j.virol.2014.01.010 sha: doc_id: 273745 cord_uid: mwjh5se7 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, 2005; lai et al., 2007) . three groups of coronaviruses have been identified, based on differences in serology and genotyping (cavanagh, 1997; spaan et al., 2005) . 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., 2007) . the host range and tissue tropism of coronaviruses depend on interactions between the viral s glycoprotein and receptors on susceptible cells (bosch et al., 2003; gallagher and buchmeier, 2001) . porcine transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhea virus (pedv) are swine-specific enteric coronaviruses that are antigenically distinguishable (lai et al., 2007; pensaert and yeo, 2006) . 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, 2006; sanchez et al., 1992) . two decades ago, porcine aminopeptidase n (papn) was identified as a cellular receptor for tgev (delmas et al., 1992) . since that time, several limited reports have showed that addition of exogenous papn facilitates cell infection by pedv (li et al., 2007; oh et al., 2003.) . recent evidence also indicates that increased papn receptor density on the surface of st cells contributes to cell infection by pedv (nam and lee, 2010) . 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 12-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., 2011a) . 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 50% of that produced in uninfected, peptide-free cells. the cc 50 values were greater than 1000 μg/ml. all subsequent antiviral experiments were performed at peptide concentrations below the experimentally-determined cc 50 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. 1a) 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 50% when the dilutions were reduced from 1:64 to 1:4. when vero cells were pre-treated with peptide (cell pretreatment assay) prior to virus infection (fig. 1b) , 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. 1c) , the results indicated that both peptides h and s inhibited pedv infectivity where ec 50 values were approximately 1 μg/ml and 62.5 μg/ml, respectively. the antiviral activity of peptide h was dose-dependent and at 250 μg/ml it exhibited greater than 95% anti-pedv activity which is significantly higher than peptides s or f (p o0.01); at 15.6 μg/ml, inhibition was greater than 70%. the selectivity indices si of peptides h and s were 1000 and 16, respectively. peptide f showed little inhibitory activity against pedv infection even at concentrations z 1000 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. 2 , 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 (1 mm/ml) (fig. 3) 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. 4) . at 31.25 μg/ml and 15.625 μg/ml, peptide h showed reduction of viral rna synthesis (po 0.05). however, at 250 μg/ml, 125 μg/ml and 62.5 μg/ml it significantly decreased viral rna synthesis (p o0.01) 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. 5 , the phage h reacted with a protein with an approximate molecular mass of 220 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 m13 phage library and two phages bearing non-selected peptides did not react with the pedv s protein (fig. 5) . 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 5 â 10 3 /ml. (c) peptides h, s or f were first incubated with pedv at 37 1c for 1 h, and then the peptide treated viruses (pfu ¼ 5 â 10 3 /ml) were used to infect vero cells at 37 1c. 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 1, 2, 3, 4, and 5 are 250 μg/ml, 125 μg/ml, 62.5 μg/ml, 31.25 μg/ml, and 15.625 μg/ml, respectively. anti-pedv antibody dilutions 1, 2, 3, 4, and 5 are 1:4, 1:8; 1:16; 1:32; and 1:64, respectively. bars show the standard deviation from three independent assays. h exhibited significantly higher inhibitory effects (po 0.01) than peptide f or pbs on the pre-attachment of pedv to vero cells ( fig. 6a ) when virus was incubated with peptide h at 4 1c for 1 h prior to incubation with the cells. when peptide h and pedv were co-incubated with vero cell at 4 1c for 1 h before shifting to 37 1c (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 40.05) than when peptide h and pedv were co-incubated with cells absent a pre-incubation step (fig. 6b) 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 37 1c absent any preincubation step, no effective inhibition of cell infection was observed (fig. 6c ). 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 ¼5 â 10 3 /ml) at 4 1c or 37 1c for 1 h, then the peptide-treated viruses were used to infect cells at 37 1c. the results showed that the inhibition rate at 37 1c was significantly higher (p o0.01) than that at 4 1c when the lower concentrations of peptide h were applied. the inhibition ratio reached 61.86% at 37 1c but only 1.25% at 4 1c at the lowest concentration (15.625 μg/ml). in contrast, high concentrations of peptide h gave rise to a similar inhibition of pedv infectivity (fig. 6d ). 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., 2011b; li and ren, 2011) . 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., 2005 carnazza et al., 2008) , fungi (bishop-hurley et al., 2002; fang et al., 2006) and viruses (ren et al., 2011a ferrer and harrison, 1999; welch et al., 2007; wu et al., 2011; yang et al., 2003) . 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, 2010; kenny and maroux, 1982; lendeckel et al., 2000) . 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., 1992; liu et al., 2009) . recently, three papn-binding peptides h, s, and f were identified using papn as an immobilized target for panning a 12-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., 2007; oh et al., 2003) . interestingly, tgev can be easily propagated in swine-originated cells such as st cells (delmas et al., 1992; hofmann and wyler, 1988) 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., 2011a; liu et al., 2009) 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 220 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 2 à δδct method. peptide concentrations 1, 2, 3, 4, 5, and 6 are 0 μg/ml, 15.625 μg/ml, 31.25 μg/ml, 62.5 μg/ml, 125 μg/ml, 250 μg/ml, respectively; line 7 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 1 μg/ml needed to achieve ec 50 . 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 37 1c 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 4 1c and internalization does not happen until the temperature is raised to 37 1c (baldick et al., 2010) . our results clearly showed that the effects of peptide h occurred during the incubation step at 4 1c rather than the 37 1c 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., 1993) 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 10% fetal bovine serum, (fbs, gibco, us), 100 units/ml of penicillin and 100 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 10 μg/ml trypsin (gibco, us) (ren et al., 2011b) . tgev strain pur46-mad was propagated in st cells (ren et al., 2008; yin et al., 2010) . porcine pseudorabies virus (prv) strain kaplan was propagated in vero cells (ren et al., 2011c) . st or vero cells were seeded in 96-well plates at a density of 5 â 10 4 cells/well and cultured in dmem containing 10% fbs at 37 1c under 5% co 2 for 24 h followed by addition of serial dilutions (62.5-1000 μg/ml) of the tested peptides. the cells were allowed to grow for 48 h at 37 1c and proliferation was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) method. briefly, the medium was removed and 10 μl of mtt solution (0.5 mg/ml, invitrogen, us) was added and incubated at 37 1c for 4 h. then 100 μl of dimethyl sulfoxide (dmso) was added and incubated for 15 min to solubilize the formazan crystals. cell survival rate was calculated as (od 490 treatment)/(od 490 control) (paeshuyse et al., 2006) . the 50% cytostatic concentration (cc 50 ) was defined as the concentration inhibiting the proliferation of (b) pedv (pfu ¼ 5 â 10 3 /ml), peptides h or f, and vero cells were co-incubated at 4 1c for 1 h, washed, then shifted to 37 1c. (c) pedv (pfu ¼ 5 â 10 3 /ml), peptides, and vero cells were co-incubated at 37 1c for 1 h then washed prior to assaying. (d) pedv was treated with various concentrations of peptides h or f at 4 1c or 37 1c for 1 h then used to infect vero cells (pfu ¼5 â 10 3 /ml) at 37 1c. peptide concentrations 1, 2, 3, 4, and 5 are 250 μg/ml, 125 μg/ml, 62.5 μg/ml, 31.25 μg/ml, and 15.625 μg/ml, respectively. anti-pedv antibody dilutions 1, 2, 3, 4, and 5 are 1:4, 1:8, 1:16, 1:32 and 1:64, respectively. plaque assays were further performed at 72 h post-infection. bars show the standard deviation from three independent assays. exponentially growing cells by 50%. non-cytotoxic concentrations of peptides (r cc 50 ) 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., 2010) . in the first method, i.e., cell post-treatment assay, vero cells were grown in 24-well plates at a density of 5 â 10 5 cells/well for 24 h. pedv at a pfu (plaque-forming unit) of 5 â 10 3 /ml was inoculated onto confluent cells for 1 h followed by removal of the medium and incubation of the infected cells with various noncytotoxic concentrations (r cc 50 ) of peptides for 1 h at 37 1c. the cells were then overlaid with 1% methylcellulose in dmem and incubated for 72 h at 37 1c followed by crystal violet staining and plaque assays as previously described with modifications (ren et al., 2011a (ren et al., , 2011d . briefly, after the medium was removed, the cells were fixed with 4% formaldehyde in pbs for 1 h at room temperature followed by staining with 1% crystal violet solution for 20 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: 100 â [1 à (treatment wells/control wells)]. average ec 50 values (concentration inducing 50% inhibition of virus replication) were calculated and the effectiveness of each peptide were expressed using the selectivity index (si) (si¼ cc 50 /ec 50 ) (paeshuyse et al., 2006; müller et al., 2007) . in the second method, i.e., cell pre-treatment assay, vero cells were first grown in 24-well plates at a density of 5 â 10 5 cells/well for 24 h then treated with non-cytotoxic concentrations of peptide for 1 h prior to incubation with virus. the peptides were removed and the cells were washed twice with pbs. pedv at a pfu of 5 â 10 3 /ml was inoculated onto the pre-treated vero cells for 1 h. after the virus was removed, the cells were overlaid with 1% methylcellulose in dmem and incubated for 72 h at 37 1c followed by plaque assays. in the third experiment i.e., virus pre-treatment, various concentrations of the peptides were mixed with pedv (pfu ¼ 5 â 10 3 /ml) at 37 1c for 1 h prior to incubation with cells. vero cells were grown in 24-well plates at 5 â 10 5 cells/well for 24 h then the peptide/virus mixture was added to the cultured cells for 1 h. after the mixture was removed and the cells washed with pbs, the cells were overlaid with 1% methylcellulose in dmem and cultured for an additional 72 h at 37 1c followed by plaque assays as described above. in parallel, pedv-neutralizing, rabbit antiserum serially-diluted 1:2 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 24-well plates and incubated at 37 1c for 24 h. indirect immunofluorescence assays (ifa) were performed (ren et al., 2011d; baldick et al., 2010) with modification. the cells were washed twice with pre-chilled pbs then fixed with 4% paraformaldehyde in pbs for 30 min at room temperature. following two washes with pbs, they were quenched with 0.1 m glycine for 5 min then blocked with 2% bsa (sigma, us) in pbs for 30 min. samples were incubated for 1 h with anti-papn polyclonal antibody (1:1500 in pbs) (liu et al., 2009) , washed three times with pbs, and incubated with fluorescein isothiocyanate (fitc) conjugated goat anti-rabbit igg (1:1500 in pbs, zhongshan, china) for 1 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 (pfu¼5 â 10 3 /ml) at 37 1c for 1 h then added to confluent vero or st cell monolayers for 1 h. the mixtures were removed and the cultured cells washed twice with pbs then incubated with 1% methylcellulose in dmem for 72 h at 37 1c. 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., 2011d) . vero cells in 6-well plates were infected with pedv (pfu ¼5 â 10 3 /ml) pretreated with different concentrations of peptide h at 37 1c for 1 h. the culture was replaced with dmem at 37 1c for 24 h then washed 3 times with pbs. the virus-containing cultures were frozen and thawed three times followed by addition of an equal volume of 20% polyethylene glycol (pge) 8000 at room temperature and incubation for 30 min. the samples were centrifuged at 12,000 rpm for 5 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 20 μl consisting of 5 μl total rna (2.5 μg), 1 μl oligo dt, 1 μl dntp (10 mm), 0.5 μl rnase inhibitor, 7.5 μl sterile water, 1 μl mlv, and 4 μl 5 â rt-pcr buffer. the mixture was incubated at 30 1c for 10 min, 42 1c for 60 min and 95 1c for 5 min. real-time pcr was performed using abi prism 7500 real-time pcr machine (applied biosystems, usa). the information regarding primers and rt-pcr products is provided in table 1 . the real-time pcr mixture included 0.5 μl (0.5 μg) of cdna template, 10 μl of sybr taq polymerase, 0.4 μl of rox pge 2, 0.5 μl (10 pmol) of each primer, and 8.1 μl of sterile water. the reactions were incubated at 95 1c for 10 s followed by 40 cycles of 95 1c for 5 s and 60 1c for 34 s. we examined virus rna levels using primers that specifically amplify a 244 bp fragment encompassing the 3' end of a small, non-structural gene (x) and the 5' end of the pedv-n gene ( table 1 ). the expression of pedv x-n in pedv-infected vero cells was normalized to that of beta-actin and taken as 100% 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 2 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 5' cactggttgggctttctatgtc pedvx-n antisense 5'tgttagtgggtacagcgttgtt 244 sense 5' ggctcagagcaagagaggtatcc β-actin antisense 5' ggtctcaaacatgatctgagtcatct 208 western blot analysis of peptide h binding to pedv the pedv (1 â 10 6 pfu/ml) was harvested from vero cells and clarified by centrifugation at 1300g for 15 min followed by ultracentrifugation at 150,000g for 1.5 h to collect the virus. the pellets were suspended in pbs, subjected to 10% sds-page then blotted to a nitrocellulose (nc) membrane. the nc membranes were blocked overnight at 4 1c with 5% non-fat dry milk in pbs. after three washes with pbs, membranes were sliced and incubated with phage h (1 â 10 11 pfu), anti-pedv s polyclonal antibody (1:1000 in pbs), m13 phage library (1 â 10 11 pfu), and control phage bearing either the peptides svsvgmkpsprp or mscndtlcllpn. the membranes were then washed with pbs and successively incubated with anti-m13 polyclonal antibody (abcam, 1:600 in pbs) and horseradish peroxidase hrp-conjugated goat anti-rabbit igg (1:1500 in pbs) at room temperature for 1 h. protein bands were visualized using 3,3'-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 5 â 10 3 /ml) at 4 1c for 1 h then added to confluent vero cells seeded in 24-well plates at 4 1c for 1 h followed by infection at 37 1c for 72 h. second, the peptides, pedv and vero cells were co-incubated at 4 1c for 1 h, after which the incubation temperature was raised to 37 1c for 72 h. third, the peptides, pedv and vero cells were co-incubated at 37 1c for 1 h then assayed without prior incubation at 4 1c. finally, peptides were pre-incubated with pedv at 4 1c or 37 1c for 1 h followed by cell infection at 37 1c for 72 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 11.5 software; p o0.05 and p o0.01 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 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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 1 and rabies virus contribution of the porcine aminopeptidase n (cd13) receptor density to porcine epidemic diarrhea virus infection identification of a putative cellular receptor 150 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-1 entry phage displayed peptides to avian h5n1 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-1) 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 (31340003 and 31372438), sponsored by chang jiang scholar candidates program for provincial universities in heilongjiang (2013cjhb002), the program for new century excellent talents in university of ministry of education of p.r. china (ncet-10-0144). key: cord-312899-ot5pvtbl 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 10 clinical isolates of sars coronavirus to selected antiviral compounds date: 2004-09-30 journal: journal of clinical virology doi: 10.1016/j.jcv.2004.03.003 sha: doc_id: 312899 cord_uid: ot5pvtbl 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 10 clinical isolates of sars coronavirus by neutralisation tests with confirmation by plaque reduction assays. interferon-beta-1a, 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 16h 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-1a 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., 2003) , 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 10th day after the onset of symptoms with subsequent clinical deterioration in 30% of the cases despite a decreasing viral load (peiris et al., 2003) . 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 10 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 10 different sars patients who satisfied the revised who criteria for sars are listed in table 1 . 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) 15 gm, chai hu (bupleurum chinense) 10 gm, ma huang (ephedra sinica) 5 gm, xing ren (prunus armeniaca) 10 gm, sheng she gao (plaster stone) 30 gm, sheng yi ren (coix lacryma-jobi) 15 gm, gua wei pi (benincasa hispida) 15 gm, jie geng (platycodon grandiflorum) 9 gm, bo he (mentha haplocalyx) 6 gm, huang qin (scutellaria baicalensis) 10 gm, sheng gan cao (glycyrrhiza uralensis) 5 gm, jin yin hua (flos lonicerae) 15 gm, and qing hao (artemisia apiacea) 15 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 2 . artesunate is not included in this table since it is already well known as an anti-malarial drug in western medicine (price, 2000) . they were extracted as we have previously reported (lu et al., 2003) . the concentrations of baicalin, glycyrrhizin, no glycyrrhizin in plasma is found after oral administration of 100 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 (1000 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 ∼1500 mg baicalin (as tablets); also can be up to ∼6000 mg baicalin (calculated from herb, assuming 30 g herb used; the herb may contain up to 20% as baicalin) ∼300 mg glycyrrhizin (as tablets) or ∼1700 mg glycyrrhizin (calculated from the herb assuming that the herb contains 5.65% glycyrrhizin) ∼2220 mg (calculated from the herb assuming that the herb contains 7.4% chlorogenic acid) serum level (after intravenous administration) chlorogenic acid, and lopinavir in the cell culture system were also monitored by hplc (lu et al., 2003) whereas the concentration of others were monitored by neutralization assays with the vesicular stomatitis virus indiana strain and a laboratory strain of influenza a h1n1.the procedures used for in vitro antiviral susceptibility testing are as follows. initial screening of all compounds against the prototype sars coronavirus strain no. 39849 was performed in 96-well microtitre plates seeded with foetal rhesus kidney-4 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 100 tcid 50 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 48 and 72 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-4 (frhk-4) and vero-e6 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 96 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 e6 and frhk-4 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 10 different patients with sars admitted to different hospitals in hk-sar showing seroconversion towards the prototype virus infected frhk-4 cell line were used in this study (table 1) . they were isolated from the lung tissue biopsy (prototype virus, m39849), urine, and nasopharyngeal aspirates. initial screening of 20 commercially available antimicrobial agents against the prototype virus grown in frhk-4 cell line did not reveal inhibitory activities for acyclovir, ganciclovir, cidofovir, foscarnet, interferon-alpha-2a, interferon-alpha-2b, amantadine, zidovudine, stavudine, nevirapine, abacavir, and ritonavir. inhibitory activities were not detectable for glycyrrhizin, artesunate and chlorogenic acid in frhk-4 cell line. glycyrrhizin was still included for further testing because this was reported to be active in vero-e6 cell lines (cinatl et al., 2003a) . further testing by neutralization tests table 3 comparison of antiviral activity of 10 compounds against 10 strains of sars-cov in frhk4 cell line, against the prototype strains (39849) with the other 9 isolates of sars coronavirus against the active compounds confirmed detectable inhibitory activities for leukocytic interferon-alpha, interferon-beta-1a, ribavirin, lopinavir, rimantadine, and baicalin. the range of their effective concentration of compound required to reduce the plaque forming unit by 50% (ec 50 ) at 48 and 72 h, and their selectivity index are shown in table 3 . when the same neutralization test on these compounds was run in vero-e6 cell line, rimandatine, glycyrrhizin, leukocytic interferon-alpha and interferon-beta were more active especially at 72 h. moreover, pre-incubation of the cell lines with these two interferons for 16 h before adding the virus markedly enhanced the inhibitory activity by three to over 100-fold. but ribavirin, lopinavir, and baicalin were less active in the vero-e6 cell line (table 3) . as for the plaque reduction assay, vero cell lines were used instead of vero e6 or frhk-4 cell lines because antiviral activity can be demonstrated for most of the agents. only interferon-beta-1a, leukocytic interferon-alpha, lopinavir, ribavirin, rimantadine, and baicalin were tested. the ec 50 of interferon-beta-1a (8 u/ml), leukocytic interferon-alpha (30 u/ml), lopinavir (6 g/ml), ribavirin (50 g/ml), rimantadine (7 g/ml), and baicalin (11 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., 2004) . 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-1a, and ribavirin. marked synergism was seen at both 72 and 96 h. the combination of leukocytic interferon-alpha (78 g/ml) and ribavirin (25 g/ml) or interferon-beta-1a (312.5 g/ml) and ribavirin (25 g/ml) were shown to be active at 96 h of incubation (table 4) . 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 7% comparing favourably with the 15% to 27% of other areas (who, 2003) . 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., 2003a,b) . using the frhk-4 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 48 h (table 3) . 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 4 to 32. upon subsequent testing with vero-e6 cell line, both leukocytic interferon-alpha and interferon-beta-1a were more active and especially after pre-incubation for 16 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., 2003b) , 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-2a and interferon-alpha-2b. this was not unexpected because this preparation of leukocytic interferon-alpha is a multi-subtype natural interferon with predominantly interferon alpha-1 and alpha-2 in contrast to the other commercial preparation with a single subtype of recombinant interferon-alpha-2. in in vitro studies, different subtypes have been found to have different antiviral activities as well as immunological effects (foster et al., 1996) . 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., 1993) . 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., 1987; lebwohl et al., 1992) . and interferon-alpha was not effective in preventing cmv infections or treating cmv pneumonia in bone marrow transplant patients (meyers et al., 1980) . 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., 1986) . 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, 1984; higgins et al., 1983; higgins et al., 1990) . besides the high cost of interferons, the high incidence of fever and flu syndrome of up to 98% 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, 2002) . although interstitial pneumonitis and bronchiolitis obliterans organising pneumonia are rare complications of prolonged use of interferons (karim et al., 2001; ogata et al., 1994) , 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 100 mg of glycyrrhizin orally was not detectable. even with a 200 mg dose of intravenous administration, the peak serum level is only 80 g/ml which is still below the ec 50 of glycyrrhizin. although an oral dose of 1.5 gm of baicalin can only achieve a serum concentration of 0.47 g/ml, intravenous administration of a 360 mg dose of baicalin in human can achieve a peak serum concentration of 74 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-1 by two mechanisms (kitamura et al., 1998; li et al., 2000) . at the level of cellular entry, baicalin can conjugate with selected chemokines such as mip-1␤ and sdf-1␣, and interfere with their capacity to activate cellular receptors ccr5 and cxcr4 respectively. these two co-recpetors are essential elements for hiv-1 infection and therefore baicalin can inhibit env-protein mediated fusion of hiv with cells expressing cd4/ccr5 or cd4/cxcr4. baicalin has also been known to inhibit hiv-1 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-1a 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-1a 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 24 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., 2004) . 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 2-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 n3) versus human recombinant ifn-alpha 2 for inhibition of hiv-1 replication in primary human monocytes different relative activities of human cell-derived interferon-alpha subtypes: ifn-alpha 8 has very high antiviral potency isolation and characterization of viruses related to the sars coronavirus from animals in southern china intranasal interferon-alpha 2 treatment of experimental rhinoviral colds intranasal interferon as protection against experimental respiratory coronavirus infection in volunteers the efficacy of intranasal interferon alpha-2a 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-1 production in vitro recombinant alpha-2 interferon gel treatment of recurrent herpes genitalis flavonoid baicalin inhibits hiv-1 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-277547-2vim1wno 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-2 date: 2011-12-28 journal: virol j doi: 10.1186/1743-422x-8-560 sha: doc_id: 277547 cord_uid: 2vim1wno 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 -2 (denv-2) in vero cell was evaluated. anti-dengue activity of these compounds was determined at different stages of denv-2 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 50 (cc(50)) to inhibitory concentration 50 (ic(50)) for each compound. results: the half maximal inhibitory concentration (ic(50)) of quercetin against dengue virus was 35.7 μg ml(-1 )when it was used after virus adsorption to the cells. the ic(50 )decreased to 28.9 μg ml(-1 )when the cells were treated continuously for 5 h before virus infection and up to 4 days post-infection. the si values for quercetin were 7.07 and 8.74 μg ml(-1), respectively, the highest compared to all bioflavonoids studied. naringin only exhibited anti-adsorption effects against denv-2 with ic(50 )= 168.2 μg ml(-1 )and its related si was 1.3. daidzein showed a weak anti-dengue activity with ic(50 )= 142.6 μg ml(-1 )when the denv-2 infected cells were treated after virus adsorption. the si value for this compound was 1.03. hesperetin did not exhibit any antiviral activity against denv-2. the findings obtained from foci forming unit reduction assay (ffura) were corroborated by findings of the qrt-pcr assays. quercetin and daidzein (50 μg ml(-1)) reduced denv-2 rna levels by 67% and 25%, respectively. there was no significant inhibition of denv-2 rna levels with naringin and hesperetin. conclusion: results from the study suggest that only quercetin demonstrated significant anti-denv-2 inhibitory activities. other bioflavonoids, including daidzein, naringin and hesperetin showed minimal to no significant inhibition of denv-2 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-1, denv-2, denv-3 and denv-4 which are transmitted to humans mainly by two species of mosquitoes, aedes agypti and aedes albopictus [1] . 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 [2] [3] [4] . there have been numerous reports on the antiviral activity of various phytochemicals against dengue viruses and these include various flavonoids [5] [6] [7] [8] . 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 [9, 10] . antiviral activities of various other flavonoids have also been reported against some viruses including human cytomegalovirus (hcmv), hsv-1, hsv-2 and some types of human adenoviruses [11] [12] [13] . 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 [14] [15] [16] . naringin on the other hand, is a flavonone glycoside found abundantly in grapefruit juice. antiviral activity of naringin were reported against hsv-1 and hsv-2 but this finding remains controversial [12, 17] . daidzein is an isoflavone found in soybeans and its antiviral activity against influenza viruses has been reported [18] . 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-2 (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 (20 mg ml -1 ) were stored at -20 c. stock solution was diluted using cell culture medium and sterilized by a syringe filter with 0.2 μm pore size (millipore, ma, usa) right before each experiment. c6/36 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 10% fetal bovine serum (gibco, ny, usa). cultured c6/36 and vero cells were incubated at 28 c and 37 c, respectively in 5% co 2 humidified chamber. at the time of virus propagation, serum concentration was reduced to 2%. dengue virus type-2 (denv-2) new guinea c strain (ngc) was propagated using c6/36 cell line and harvested after cpe presentation on day seven post-infection. after titration, viral stock was stored at -70 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 96-well cell culture microplates were treated with different concentrations of each compound in triplicate. the treated cells were incubated for four days at 37 c followed by the addition of 15 μl of mtt solution to each well. the microplate was incubated at 37 c for 4 h. then, 100 μl of the solubilisation/stopping solution was added to each well. the optical density (od) of wells was measured at 570 using 96-well plate reader (tecan, mannendorf, switzerland). dose-response curves were plotted using graph pad prism 5 (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, 5 h before virus infection. after 5 h of pre-infection treatment, the cells were washed twice with sterile pbs and then 200 ffu of denv-2 was inoculated to the cells and incubated at 37 c for 1 h. to determine the effects of continuous treatment, different concentrations of each compound were added to the vero cells, 5 h pre-infection and continuously for 4 days post-infection. in a separate experiment, antiviral activity of compounds against intracellular replication of denv-2 was performed by inoculation of 200 ffu of virus to each well in triplicate. after adsorption of virus to the cells for 1 h at 37 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 4 days of incubation at 37 c. denv rna was then quantified using quantitative rt-pcr. in another experiment, vero cells at 80% confluency were infected with 200 ffu of denv-2 in the presence or absence of different concentrations of each compound. the microplate was kept at 37 c for 1 h for virus adsorption. then the cells were washed two times by sterile pbs and incubated at 37 c for four days. direct virucidal effects of the bioflavonoids were investigated by incubating denv-2 suspension containing 200 ffu with an equal volume of the different concentrations of each compound for 2 h at 37 c. then, vero cells were infected with the treated viral suspension in triplicate. after 1 h adsorption at 37 c, cells were washed twice with pbs in order to remove unabsorbed viruses. then the microplate was incubated at 37 c for 4 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 24 wells cell culture microplate. then the infected cells treated using different procedures described above were overlaid with 1.5% 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 [19] . the numbers of denv-2 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) × 100/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-2 genomic rna copies based on a method described previously with some modifications [20] . briefly, intracellular and extracellular denv-2 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 1 μl of extracted denv rna to the sensimix sybr green reagent (quantace, watford, united kingdom) which contained 7.4 μl ddh2o, 10 μl 2x sensimix one-step, 0.4 μl 50x sybr green solution, 10 units of rnase inhibitor, 50 pmol of forward (dnf) and also reverse (d2r) primers [21] . 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 50°c for 30 min, initial denaturation at 95°c for 10 min, followed by 45 cycles of 95°c for 15 sec, 59°c for 30 sec and 72°c for 30 sec. melting curve analysis was subsequently performed at temperature from 60°c to 98°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-2 stock with known titer. graph pad prism for windows, version 5 (graph pad software inc., san diego, ca, 2005) was used to determine the cytotoxic concentration 50 (cc 50 ) and inhibitory concentration 50 (ic 50 ) values of bioflavonoids. selectivity index value (si) was determined as the ratio of cc 50 to ic 50 for each compound. mtt assay was used to determine cytotoxicity of each bioflavonoid on vero cells and the cc 50 value of each compound was calculated (table 1 and figure 1 ). vero cells were treated by bioflavonoids for 4 days which was the same duration used for antiviral activity assay. results showed that hesperetin with cc 50 = 110.3 ± 0.32 μg ml -1 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 50 252.6 ± 0.17 and 147.8 ± 0.31 μg ml -1 , respectively. meanwhile, naringin with cc 50 = 230.3 ± 0.19 μg ml -1 showed the least cytotoxic effects against vero cells. cells treated with vehicle control, 1% dmso did not show any cytotoxicity against vero cells. results of vero cells pre-treatment with the compounds showed that 50 μg ml -1 of quercetin could decrease the number of denv-2 foci by 14% ± 1.5 when compared to the non-treated cells. however, there was no prophylactic activity against denv-2 from other compounds (data not shown). in post-adsorption assay, quercetin exhibited the most significant antiviral activity against denv-2 amongst the bioflavonoids tested with ic 50 = 35.7 μg ml -1 (figure 2a) . the si value for quercetin in post-adsorption assay was relatively high at 7.07. it was also demonstrated that the level of denv-2 specific rna production in the presence of 50 μg ml -1 of quercetin decreased by more than 67% ± 1 when compared to the non-treated infected cells (figure 2b ). daidzein showed very weak anti-dengue activity with ic 50 = 142.6 μg ml -1 when the infected cells were treated after denv-2 adsorption (figure 2a ). its related si value was 1.03. the levels of denv-2 rna production in the presence of 50 μg ml -1 of daidzein decreased by only 25.3% ± 0.7 when compared to the non-treated infected cells (figure 2b ). naringin and hesperetin did not exhibit any anti-dengue activity when they were used after adsorption of denv-2 to the vero cells ( figure 2 ). although there was no significant direct virucidal activity against denv-2 by quercetin, continuous treatment of cells from 5 h before virus infection up to 4 days post-infection exhibited anti-dengue activity with ic 50 = 28.9 μg ml -1 (figure 3a) . the si value for continuous treatment with quercetin was 8.74 and higher than the si value (7.07) for post-adsorption assay. in addition, the level denv-2 rna production decreased by more than 75.7% ± 1.57 when vero cells were treated with 50 μg ml -1 of quercetin, 5 h before virus infection and up to 4 days post infection ( figure 3b ). there was no significant change in the antiviral activity of daidzein when cells were treated continuously from 5 h before virus infection up to 4 days post infection comparing to its anti-dengue activity for postadsorption treatment (figure 1 ). no significant antiviral activity for naringin and hesperetin was observed for the continuous treatment against denv-2 ( figure 2 ). however, naringin exhibited anti-adsorption activity when it was added to the cells at the same time of virus adsorption. the ic 50 value for naringin was 168.2 μg ml -1 and its related si was 1.3 ( figure 3a ). there was a reduction of 25.8% ± 0.76 in denv-2 rna production in the presence of 50 μg ml -1 of naringin (figure 4a) . the majority of the viral foci in cells treated with 50 μg/ml quercetin appeared smaller, less intensely stained and more diffused within the focus (figure 5b) , compared to the larger, well-defined and more intensely stained foci of the untreated cells (figure 5a ). 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-2 for all the tested figure 4 anti-adsorption activity of flavonoids against denv-2. flavonoids were added directly to virus inocula for 2 h at 37 c. the inocula were used to infect vero cell monolayers in 24 wells cell culture microplates. the reduction in foci forming unit was calculated relative to the controls maintained in parallel (a) and the respective denv-2 rna copy numbers were quantified using qrt-pcr (b). data from triplicate experiments were plotted using graph pad prism version 5 (graph pad software inc., san diego, ca). the reduction of intracellular replication of dengue virus by 76.9% and 75% following treatment with 25 μm of glabranine and 7-o-methyle glabranine, respectively [22] . similarly, other synthetic flavonoid derivatives also showed antiviral activity in hepg2 cells [23] . whereas, pinostrobin was reported to inhibit denv-2 ns2b/ns3 protease an enzyme important in dengue virus replication in an in vitro study [24] . 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 [22, 23] , 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-2 in vero cells. selectivity indices for quercetin when the infected cells were treated or when uninfected cells were treated continuously 5 h before infection until 4 days post-infection were 7.07 and 8.74, 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-2 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 50 = 3.2 μm [13] . quercetin was also reported to be effective against herpes viruses where it is more specific against hsv-1 with si = 22 compared to hsv-2 with si = 5.7 [11] . 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 [25, 26] . sylimarin, a flavonoid found effective against hepatitis c virus (hcv), another member of flaviviridae family [27] , 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-2 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-1 and hsv-2 are inconclusive [12, 17] . in addition it was reported that naringin did not exhibit any antiviral activity against another rna virus, sindbis virus [14] . 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 50 = 168.2 μg ml -1 ) and its related selectivity index (si = 1.3), it may not be a good candidate for further development as anti-dengue drug. similarly, daidzein activity against denv-2 was not significant compared to quercetin (si = 1.03). continuous treatment of the infected vero cells from 5 h before virus infection up to 4 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 [14] . therefore, hesperetin is also not recommended for further investigations for anti-dengue drug development. in all our experiments, we showed that 0.5% of dmso, the highest concentration of solvent used in the bioflavonoid treatment did not exhibit any antiviral activity against denv-2 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 [28] . while the flavonoids in general share common basic molecular base structure, flavone (2-phenyl-1,4-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 [28] , 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-3-o-rutinoside) showed no significant inhibition properties [28] . 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 [29] [30] [31] . 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 [32] [33] [34] . the other topic of research would be combination drug study. at its current calculated ic 50 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 [35] . 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 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emulsifiers enhances the absorption of orally administered 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-2 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-343132-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: 2020-07-15 journal: front microbiol doi: 10.3389/fmicb.2020.01578 sha: doc_id: 343132 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 16s 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 ypk30 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 ypk30 possessed in vitro prophylactic, therapeutic, and direct-inhibitory effects against pedv in the vero cell model. the expression levels of type 1 interferon (ifn)-dependent genes, including myxovirus resistance 1 (mx1) and interferon-stimulated gene 15 (isg15), were significantly increased after treatment with intracellular extracts of ln. mesenteroides ypk30 for 24 h. such expression suggests that the anti-pedv activity of ln. mesenteroides ypk30 could be attributed to its up-regulatory effect on the expression of mx1 and isg15 genes. these results suggested that ln. mesenteroides ypk30 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 2 (pcv2), and transmissible gastroenteritis virus (tgev; meng, 2012; gerdts and zakhartchouk, 2017) . 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, 2007) . 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, 2015) . 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 50-90% in neonatal piglets, which has resulted in huge economic losses to the swine industry worldwide (koonpaew et al., 2019; wang et al., 2019) . 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., 2019; koonpaew et al., 2019) . 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., 2019) . 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., 2012; alonso and guarner, 2013) . 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., 2015; yadav and shukla, 2019) . 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, 2017; azad et al., 2018) . those immunoregulatory probiotics provide host protection against pathogenic infections by modulating innate and adaptive antiviral immune responses (villena et al., 2016) . several probiotic strains, most of them belonging to lactobacillus and bifidobacterium genera, have been shown to perform antiviral activities (villena et al., 2016; arena et al., 2018) . 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., 2015; villena et al., 2016; arena et al., 2018; eguchi et al., 2019) . 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., 2014) . 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., 2016) . 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, 1985) . 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., 1999; wang et al., 2012; bourrie et al., 2016) . 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., 2015) . 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 1 ifn-dependent genes, including 2'-5'-oligoadenylate synthetase 1 (oas1), myxovirus resistance 1 (mx1), and interferon-stimulated gene 15 (isg15) in vero cells. a total of 29 lab strains were isolated from kefir grains according to the method described by lin et al. (1999) . the lab isolates were cultured in de mann, rogosa, sharpe (mrs) broth (oxoid, basingstoke, uk) at 37°c for 16 h without shaking. the bacterial concentrations were measured by optical density readings at 600 nm or by traditional colony counting methods (vinderola et al., 2000) . 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 37°c for 24 h and then observed. for cell morphological observation, the lab isolates were cultured in mrs broth (oxoid) at 37°c for 16 h without shaking. the bacterial cells were harvested by centrifugation at 5,000g for 20 min at 4°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. (1990) and naser et al. (2005) . genomic dna of the bacterial isolates was isolated using the dneasy blood & tissue kit (qiagen inc., valencia, ca, usa). the standard 16s ribosomal rna (rrna) gene primers and rna polymerase α subunit gene rpoa primers were used for pcr to amplify the 16s rrna gene and rpoa gene sequences of the bacterial isolates, respectively (weisburg et al., 1990; naser et al., 2005) . 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 16s 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 50 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 10°c, 37°c, ph 4.8, or in the presence of 10% ethanol according to the methods described by lin et al. (1999) . 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 (1983) . the vero c1008 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 37°c in a humidified atmosphere of 95% air and 5% co 2 in dulbecco's modified eagle's medium (dmem, gibco, grand island, ny, usa) supplemented with 10% fetal bovine serum (fbs; moregate biotech., queensland, australia). for evaluation of cytotoxicity, a 1.0-ml aliquot of the 16-h culture of each bacterial strain was centrifuged at 9,000g for 10 min at 4°c. the bacteria were collected, washed twice with sterile phosphate-buffered saline (pbs; 0.1 m, ph 7.0), resuspended in 1.0 ml of sterile pbs, and sonicated for 10 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 13,000g for 20 min at 4°c. the intracellular extracts were harvested, and the cytotoxicity on vero cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (mtt) assay according to the method described by mosmann (1983) . briefly, vero cells were seeded at a density of 1.5 × 10 5 cells/well on a 24-well plate in 500 μl of dmem. after incubating at 37°c for 24 h, 100 μl of the bacterial intracellular extracts were added into each well and incubated at 37°c for another 24 h. after washing twice with sterile pbs, the cells were incubated with 500 μl of mtt (5 mg/ml in pbs) at 37°c for 2 h. after the incubation, the medium was removed, and 200 μl of dimethyl sulfoxide (dmso) were added into each well to dissolve the formazan crystals. the absorbance was measured at 570 nm using a microplate reader (victor 3 , 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 52 strain was isolated in early 2014 from the intestinal homogenate of a 7-day-old suckling pig in taiwan and adapted to vero cells as previously described by chang et al. (2017) . viral infection and propagation were performed in vero cells according to the method described by chang et al. (2017) . before the anti-pedv activity screening experiments were conducted, the viral titers of pedv were adjusted to 200 fifty-percent tissue culture infective dose (tcid 50 )/ml, and the intracellular extracts of bacterial isolates were prepared as described above. vero cells were seeded at a density of 3 × 10 4 cells/well on a 96-well plate in 100 μl of modified postinoculation (pi) medium containing dmem (gibco, grand island, ny, usa) supplemented with tryptose phosphate broth (0.3%), yeast extract (0.02%), and 10 μg/ml of trypsin. after incubating at 37°c for 24 h, 100 μl of the bacterial intracellular extracts were added into each well and incubated at 37°c for another 24 h. after washing the cells twice with pi medium, 200 μl of pi medium containing 200 tcid 50 /ml of the pedv was added into each well and incubated at 37°c for 1 h. after 1 h of incubation, the supernatants were replaced by fresh pi medium and the cells were maintained at 37°c for 48 h. after washing twice with sterile pbs (0.1 m, ph 7.0), 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 1.0-ml aliquot of the 16-h culture of each lab strain was centrifuged at 9,000g for 10 min at 4°c. the resultant extracellular supernatants and bacterial cells were harvested separately. the bacterial cells were washed twice with sterile pbs, resuspended in 1.0 ml of sterile pbs, and sonicated for 10 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 13,000g for 20 min at 4°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 96-well plate and incubated at 37°c for 24 h. afterward, the cells were washed with pi medium, and 200 μl of pi medium containing 200 tcid 50 /ml of pedv were added into each well and incubated at 37°c for 1 h. after 1 h of incubation, the supernatants were replaced by 100 μl of fresh pi medium and 100 μl of the bacterial intracellular extracts. the cells were maintained at 37°c for 48 h. after washing twice with sterile pbs, the cell metabolic activity was determined by mtt assay as described previously. . glyceraldehyde 3-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 1 . the data were analyzed using spss version 25 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 0.05 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 0.05 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 16s rrna and rpoa gene sequence analysis, three isolated strains belong to the species enterococcus durans, 16 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 2 ). 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 24 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-α2b was used as a positive control. vero cells pretreated with ifn-α2b prior to pedv infection showed a significantly higher cell metabolic activity and less cytopathic effects than the un-pretreated cells (figures 1, 2) , indicating that ifn-α2b 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 1 ). in addition, vero cells pretreated with the intracellular extracts of ln. mesenteroides also showed less cytopathic effects than the un-pretreated cells (figure 2) , 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 3 , 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-α2b (p > 0.05) but were significantly higher than the un-pretreated cells (p < 0.05), 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 ypk30 intracellular extract, cell-wall fraction, and extracellular supernatant against pedv among the ln. mesenteroides strains, a strain designated ypk30 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 ypk30 against pedv. as shown in figure 4 , the metabolic activity of vero cells pretreated with the intracellular extracts and extracellular supernatants of ypk30 were similar to each other and were significantly higher than those of un-pretreated cells or those pretreated with the cell-wall fractions of ypk30 (p < 0.05), indicating that both of the intracellular extracts and extracellular supernatants of ypk30 possessed in vitro prophylactic effects against pedv. vero cells were infected with pedv for 1 h, and then the remaining pedv was removed. the pedv-infected cells were treated with the intracellular extracts of ypk30 or ifn-α2b for 48 h, and then the metabolic activities of vero cells were determined. as shown in figure 5 , pedv-infected vero cells treated with the intracellular extracts of ypk30 for 48 h showed higher cell metabolic activity than the untreated cells or those treated with ifn-α2b (p < 0.05), indicating that the intracellular extracts of the ypk30 strain possessed an in vitro therapeutic effect against pedv. vero cells were co-incubated with pedv and the intracellular extracts of ypk30 or ifn-α2b for 1 h. after removal of the pedv and intracellular extracts of ypk30, the vero cells were incubated for 48 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 ypk30 and pedv simultaneously were significantly higher than those treated with pedv alone or those treated with pedv and ifn-α2b (figure 6 ; p < 0.05). these results suggested that the intracellular extracts of ypk30 possessed an in vitro direct-inhibitory effect against pedv in vero cells. in order to elucidate the antiviral mechanisms of the ypk30 strain, the expression levels of type 1 ifn-dependent genes, including oas1, mx1, and isg15, were quantified in vero cells after 0, 24, or 48 h of treatment with the intracellular extracts of ypk30. as shown in figure 7 , ifn-α2b, which served as the positive control, significantly increased the expression levels of oas1, mx1, and isg15 genes in vero cells at 24 and 48 h. the intracellular extracts of ypk30 also significantly increased the expression levels of mx1 and isg15 genes but did not affect the expression levels of the oas1 gene in vero cells at 24 h. however, the expression levels of oas1, mx1, and isg15 genes in vero cells did not differ between the untreated and ypk30-treated groups at 48 h. according to the 16s rrna and rpoa gene sequence analysis, ypk30 exhibited 99.93 and 99.57%, respectively, identity with ln. mesenteroides (table 2) , and its 16s rrna and rpoa gene sequences were deposited in the ncbi genbank database under accession number mt293805 and mt333858, respectively. macroscopic observation showed that ypk30 exhibited a smooth and grayish white colony morphology and did not possess hemolytic capacity on blood agar plate. the cells of ypk30 appeared purple after gram staining, indicating the strain ypk30 was gram positive. microscopic observation showed the cells of ypk30 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 10 or 37°c but not growth at ph 4.8 or in 10% ethanol) indicated that ypk30 was closely related to species ln. mesenteroides. to further confirm the identification of ypk30 with the species ln. mesenteroides, the biochemical characteristics of ypk30 were compared with those of ln. mesenteroides subsp. cremoris atcc 19254, ln. mesenteroides subsp. dextranicum atcc 19255, and ln. mesenteroides subsp. mesenteroides atcc 8293 by using the api 50 ch system. analysis of carbon all data are expressed as mean ± sd (n = 3). bars marked with a star or double stars mean that they are significantly different from the control (cells treated with pbs) at the 5 or 1% confidence level, respectively. source utilization profiles indicated that both ypk30 and ln. mesenteroides subsp. dextranicum atcc 19255 grew on six out of 49 carbohydrates, including n-acetyl glucosamine, d-fructose, d-glucose, d-mannose, saccharose, and d-trehalose. distinct variation was observed between ypk30 and ln. mesenteroides subsp. cremoris atcc 19254 in the metabolism of the sugars d-fructose, d-mannose, and d-trehalose. additionally, distinct variation was observed between ypk30 and ln. mesenteroides subsp. cremoris atcc 19254 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 ypk30 were similar to those of ln. mesenteroides subsp. dextranicum. according to the results of microscopic observations, biochemical characteristics, and the 16s rrna and rpoa gene sequence analysis, the features of ypk30 were consistent with those of ln. mesenteroides subsp. dextranicum, as described in bergey's manual of systematic bacteriology (vos et al., 2009) . the vero cell line is one of the most commonly used cell lines for pedv isolation and propagation (koonpaew et al., 2019) . 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 1) . in addition to the in vitro prophylactic effect, the intracellular extracts of ln. mesenteroides ypk30 also possessed in vitro therapeutic effect and in vitro direct-inhibitory effects against pedv in vero cells (figures 4-6) . vero cells have a major deletion in the type 1 ifn gene cluster, which results in ifn deficiency (desmyter et al., 1968; koonpaew et al., 2019) . although vero cells do not secrete type 1 ifns when infected by viruses, they still have the type 1 ifn receptors and respond normally to type 1 ifns. therefore, vero cells were widely used to compare virus-mediated ifn antagonism specific to the ifn signaling pathway (simmons et al., 2010) . in the in vitro prophylactic and therapeutic models, the intracellular extracts of ln. mesenteroides ypk30 did not directly interact with pedv by physical contact. therefore, the in vitro prophylactic and therapeutic effects of the intracellular extracts of ln. mesenteroides ypk30 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 ypk30 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., 2018; biliavska et al., 2019) . 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., 2010; kanauchi et al., 2018) . 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., 2018; mousavi et al., 2018) . in addition, specific probiotic bacteria could synthesize some antiviral metabolites, such as lactic acid, hydrogen peroxide, or bacteriocins (al kassaa et al., 2014) . in the present study, the intracellular extracts of ln. mesenteroides ypk30 possessed an in vitro direct-inhibitory effect against pedv in vero cells (figure 6) . future studies will be aimed at identifying the mechanisms of the in vitro direct-inhibitory effect of the intracellular extracts of ln. mesenteroides ypk30 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 mx1 and rnase l in porcine intestinal epithelial cells (kanmani et al., 2018) . 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., 2015) . in the present study, pretreatment of vero cells with the extracellular supernatants or intracellular extracts of ln. mesenteroides ypk30 for 24 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 ypk30 possessed in vitro prophylactic effects against pedv in vero cells. however, pretreatment of pedv cells with the cell-wall fractions of ln. mesenteroides ypk30 for 24 h before infection with pedv did not impede pedv replication. according to these observations, we suggested that the immunomodulatory activity of ln. mesenteroides ypk30 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 1 ifns exert their antiviral activities though the induction of hundreds of isgs (lenschow et al., 2005) . classical isgs responsible for inhibition of viral infection include oas1, mx1, and isg15 (schoggins, 2014) . oas1, which belongs to the oas enzyme family, is activated by double-stranded rna binding, catalyzes the formation of 2′-5′ oligoadenylates to activate cellular rnase l, which in turn, degrade cellular and viral rna (choi et al., 2015) . mx1 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, 2014) . isg15 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, 2014) . in the present study, we determined the effects of intracellular extracts of ln. mesenteroides ypk30 on the expression levels of oas1, mx1, and isg15 genes in vero cells and found that treatment of vero cells with the intracellular extracts of ln. mesenteroides ypk30 did not affect the expression levels of the oas1 gene but significantly increased the expression levels of mx1 and isg15 genes 24 h after treatment (figure 7) , indicating that the anti-pedv activity of the intracellular extracts of ln. mesenteroides ypk30 could be attributed to its up-regulatory effect on the expression of mx1 and isg15 genes in vero cells. according to the results of microscopic observations, biochemical characteristics, and the 16s rrna and rpoa gene sequence analysis, ypk30 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, 2011) . 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., 2016) . several ln. mesenteroides strains are reported to have anti-listerial, antiviral, or immunomodulatory activities (seo et al., 2012; shao et al., 2020) . 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, 1994; holland and liu, 2011; arakawa et al., 2016) , and some exopolysaccharides produced by ln. mesenteroides showed antiviral and immunomodulatory activities (nácher-vázquez et al., 2015; mahdi et al., 2019) . in our study, we found that the intracellular extracts of ln. mesenteroides ypk30 possessed in vitro prophylactic, therapeutic, and direct-inhibitory effects against pedv in vero cells, which occur in part through the up-regulation of mx1 and isg15 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 ypk30 against pedv infections in piglet infectious challenge models. the lab strain ypk30 isolated from kefir grains was genotypically and phenotypically characterized as belonging to ln. mesenteroides subsp. dextranicum. ln. mesenteroides subsp. dextranicum ypk30 displayed in vitro prophylactic, therapeutic, and direct-inhibitory effects against pedv in vero cells via up-regulation of mx1 and isg15 expression in vero cells. these findings suggest that ln. mesenteroides subsp. dextranicum ypk30 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 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interferon-stimulated genes: roles in viral pathogenesis evaluation of leuconostoc mesenteroides yml003 as a probiotic against low-pathogenic avian influenza (h9n2) virus in chickens the probiotic, leuconostoc mesenteroides, inhibits listeria monocytogenes biofilm formation short communication: antiviral activity of porcine ifn-λ3 against porcine epidemic diarrhea virus in vitro a determinant of sindbis virus neurovirulence enables efficient disruption of jak/stat signaling bacteriocins produced by leuconostoc species intestinal innate antiviral immunity and immunobiotics: 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 16s 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 108-2313-b-002-012 and most 108-2321-b-002-064) and academia sinica (grant nos. as-kpq-108-itar-td03 and as-kpq-109-itar-td03). key: cord-355440-20yq6zj0 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: 2006-09-06 journal: eur j immunol doi: 10.1002/eji.200535587 sha: doc_id: 355440 cord_uid: 20yq6zj0 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 e6 cells, while pretreatment of free virions with snap only had a limited effect on their viability. in contrast, 3‐morpholinosydnonimine hydrochloride (sin‐1), a peroxynitrite donor, inhibited virus replication only to a very low extent in vitro, but pretreatment of virus with sin‐1 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 [1] . 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 [2] . the pathogenesis in man is only poorly understood, but immune-mediated mechanisms have been suggested [3, 4] . 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 1, vaccinia virus, epstein-barr virus, influenza virus and sars coronavirus [5] [6] [7] . 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 2 -) together form peroxynitrite (onoo -), and other reactive nitrogen intermediates (rni) [8] . 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 [9] . 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 [5, 10] , due to oxidation and nitration of cellular lipids, dna and proteins [8] . 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 [11] . influenza virus pneumonia [12] and neurotropic virus infections are other diseases where no is believed to contribute to the pathogenesis [10] , and we have detected elevated levels of no production in suckling mice that succumbed to hantavirus infection [13] . 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 [14] , 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 [15] [16] [17] , as well as in monkeys infected with puumala hantavirus (puuv) [18] . in contrast, infection of the natural host peromyscus maniculatus with sin nombre hantavirus (snv) does not induce no production [15] . the elevated levels of rni detected in hfrs/hcps patients have been suggested to play a part in the pathogenesis [15] . 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) [19] . two forms of nos, neuronal nos (nnos or nos1) and endothelial nos (enos or nos3) are constitutively expressed, whereas inos (or nos2) is strongly induced by cytokines and other immunoregulatory stimuli [20] . in the present study, we investigated the effect of no and peroxynitrite on hantavirus replication in vero e6 cells and on free mature virions by using s-nitroso-nacetylpenicillamine (snap; an no-donor), 3-morpholinosydnonimine hydrochloride (sin-1; 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 1 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 a549, the monkey kidney epithelial cell lines vero and vero e6 8 days post infection, from the human hepatoma cell line huh-7 5 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 4 days post infection. the viability of htnv-infected cells treated with 100 lm snap or 100 lm of the control n-acetylpenicillamine (nap), or medium alone, starting 12 h before infection and then replenished every 12 h, was examined. no toxicity of snap could be measured using an mtt test 54 h post infection (data not shown), showing that 100 lm snap is not toxic for vero e6 cells. the nitrite concentration in medium measured 12 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. 1) . as expected, no nitrate was detected in medium from cells incubated with nap for 12 h (fig. 1) . whether no had an effect on htnv replication was then tested. htnv-infected cells were incubated with 6.25-100 lm snap or nap, starting 1 h after infection. the media were subsequently changed every 12 h, and supernatants were drawn at 30 h post infection and titrated. no viable viruses were detected in supernatants from cells treated with 100 lm snap ( fig. 2a) . from cells treated with 50 lm snap, 85% 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. 2a , 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 100 lm snap, 100 lm nap, or medium alone from 1 h post infection. a complete inhibition of released virus was observed for puuv at 54 h post infection, and for dobv at 30 h post infection, whereas an approximately 90% inhibition was detected for saav at 30 h post infection (fig. 2b ). since it has been shown that replication of vesicular stomatitis virus is more efficiently inhibited when the cells are pretreated with exogenous no donors [21] , we compared treatment of vero e6 cells with 100 lm snap starting 12 h before, 1 h after, or 12 h after htnv infection. the media were changed every 12 h, and supernatants were sampled 30 h post infection. no difference was observed between adding snap 12 h before infection or 1 h post infection (fig. 2c) , showing that pretreatment is not needed for no-induced inhibition of replication. however, a stronger inhibition was observed when snap was added 12 h before or 1 h post infection, as compared to 12 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 1 wk (at this time point all cells were infected; data not shown). cells were then treated with 12.5-100 lm snap. at 30 h after the initial treatment with 50 and 100 lm snap, the titers of released virus were approximately 80 and 87.5% lower, respectively, as compared to cells treated with nap or medium alone (fig. 2d ). lower concentrations of snap had no effect on the virus titers (fig. 2d , and data not shown). we have previously shown that il-1b together with ifn-c up-regulates inos expression in vero e6 cells [7] . here, we further examined the effect of cytokineinduced no on hantavirus replication in vitro. treatment of cells with il-1b (10 ng/ml) alone had no effect on htnv replication (data not shown), whereas ifn-c (400 u/ml), as reported earlier [22] , inhibited the viral replication (data not shown). no increased level of nitrite was detected in cells treated with il-1b or ifn-c alone (data not shown), suggesting that the expression of inos in vero e6 cells requires both il-1b and ifn-c. approximately 20 lm of nitrite was detected in the medium 48 h after stimulation with il-1b and ifn-c. to test the effect of cytokine-induced no on htnv replication, cells were infected with htnv. after 1 h, cells were stimulated with il-1b and/or ifn-c in the presence of 1 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 24 h post infection. supernatants were collected 48 h post infec-tion for virus titration. the viral titers in supernatants from cells stimulated by il-1b combined with l-nmma or d-nmma showed no clear differences (approximately 6% more viruses in l-nmma + il-1b-treated cells compared to d-nmma + il-1b-treated cells). similarly, l-nmma or d-nmma had no clear effect on the viral titers from cells stimulated by ifn-c (approximately 5% 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 260% 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-1b together with ifn-c (fig. 3) . thus, cytokine-induced no can inhibit htnv replication in vero e6 cells. we further tested if no affected the levels of n protein expressed after infection of vero e6 cells. cells were treated with 100 lm snap, 100 lm nap, or medium alone, with start at 1 h post infection, and then media were changed every 12 h. treatment with 100 lm snap had no effect on the total cellular protein, or b-actin, levels in vero e6 cells ( [7] , and data not shown). in samples drawn 30 h post infection, htnv n protein was detected in nap-treated and untreated cells, but not in snap-treated cells (fig. 4a) . 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 100 lm snap, 100 lm nap or normal medium, as described above. the levels of viral rna in the cells 30 h post infection were analyzed. approximately 85% less viral rna was detected in snap-treated cells as compared to the nap-or medium-treated cells (fig. 4b) . as peroxynitrite was recently shown to have antiviral capacities [9] , we then investigated if hantavirus replication was sensitive to treatment with peroxynitrite. in medium with 100 lm sin-1, 66 lm nitrite was detected 12 h after incubation, showing that most of the added sin-1 had decayed (data not shown). to vero e6 cells, 100 lm sin-1 was added after infection with htnv, and fresh medium containing sin-1 was added at 12 h and 24 h after infection. at 30 h post infection, the supernatant was collected and titrated. approximately 40% less viable virus were observed in cells incubated with 100 lm sin-1, as compared to controls (fig. 5) . to test if no or peroxynitrite had a direct inhibitory effect on free mature virions, htnv was incubated with 1 lm to 1 mm snap, 1 lm to 1 mm sin-1, 1 mm nap, or with normal medium for 4 days at +4 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-1 (data not shown). no nitrite was detected after addition of 1 mm nap to the media (data not shown). the virus was 1000fold diluted before titration to rule out the effect of potential inhibition of viral replication by residual snap or sin-1. while 10 lm sin-1 inactivated approximately 75% of the virus, 100 lm sin-1 inactivated more than 90% of the virus, and 1 mm sin-1 almost all (fig. 6 ). approximately 25% reduction in viability was observed for 1 mm and 100 lm snap, as compared to the nap or medium controls (fig. 6) . to test if no has a role in the antiviral defense in vivo, we infected suckling c57bl/6 mice (inos -/and inos +/+ controls) with 5000 focus forming units (ffu) dobv, previously shown to induce no production in and to be lethal for suckling mice [13] . all mice in the two groups (inos -/-, n = 6; controls, n = 6) showed ruffled fur, paralysis of the limbs, and progressively diminishing mobility, at day 15 after infection, and were sacrificed at higher levels of dobv were observed in the inos -/-(249 593 ae 370 463 ffu/g brain) compared to the control (53 688 ae 55 172 ffu/g brain) mice (fig. 7) , 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 2 in the medium and form peroxynitrite. and although most of the no and o 2 produced by sin-1 will immediately form peroxynitrite, some no will be produced that might not react with o 2 -. 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-1 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-1, 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-1, 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 e6 cells. furthermore, davis and coworkers recently reported clearly elevated levels of nitrate/nitrite in hcps patients [15] , and groeneveld and coworkers showed the same for hfrs patients [16] . 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 3b [23] . (ii) enhanced mutation rate: no has been shown to enhance the mutation rate of another rna virus, the sendai virus [24] . (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 [21, 25] . peroxynitrite has been reported to inhibit coxsackievirus rna entry into host cells [9] . 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 [10] . direct up-regulation by virus has been shown for respiratory syncytial virus, human immunodeficiency virus, and hepatitis c virus infection [26] [27] [28] [29] . 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 [3] . rni has been suggested to be involved in hantavirus pathogenesis [15] . elevated levels of no have been detected in man [15] [16] [17] and monkeys [18] , 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 [13] . furthermore, we also observed replicating virus in saav-inoculated mice 34 days after infection, whereas mice that survived dobv infection had no replicating viruses in the brain [13] . together with the finding that inos -/suckling mice showed higher titers of replicating virus in the brain compared to normal c57bl/6 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 [9] . the viruses used were the vero e6 cell line-adapted htnv, strain 76-118 [30] , dobv, strain slovenia [31] , saav [32] , and puuv, strain kazan e6 [33] . propagation and titration of the viruses were performed on vero e6 cells [vero c1008; american type culture collection (atcc), manassas, va], as described [34] . the cells used were a549, hela, hl, huh-7, huvec (clonetics, biowhittaker, walkersville, wv), monomac (kindly provided by sa björndal, swedish institute for infectious disease control, solna, sweden), vero and vero e6. a549, hela, hl, huh-7, vero, and vero e6 cells were grown in emem supplemented with 2% fcs, 100 u/ml penicillin, 100 lg/ml streptomycin, and 1.5 g/l bicarbonate (sigma, st. louis, mo), huvec in egm-2-mv medium supplemented with egm-2-mv singlequots (clonetics), and monomac in rpmi 1640 supplemented with 10% fcs, 100 u/ml penicillin and 100 lg/ml streptomycin. suckling c57bl/6 and c57bl/6 inos -/mice, were purchased from mtc, breeding unit, karolinska institutet, stockholm, sweden. suckling mice were inoculated intracerebrally with 20 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 -70 c until further use. hearts were stored at -70 c with pbs for 24 h; after thawing, the supernatant was transferred to a new tube and used for the detection of hantavirus-specific antibodies [13] . 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-1b and ifn-c were purchased from peprotech (london, uk). sin-1, 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 2% hepes, 2% fcs, 100 u/ml penicillin and 100 lg/ml streptomycin, and incubated on confluent vero e6 cells in 24-well plates. after 1 h of incubation, cells were overlaid with 0.5% agarose-medium and incubated for a further 6-9 days, depending on the virus, at 37 c, 5% co 2 . 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 3,3 0 ,5,5 0 -tetramethylbenzidin (sigma) and counted [33] . confluent vero e6 cells grown on 24-well plates were washed, and medium alone or medium containing sin-1, snap or nap was added. cells were infected with 1000 ffu of hantavirus, corresponding to 0.005 moi. media, with or without chemicals, were changed every 12 h. supernatants were collected 6 h after the last treatment and were subsequently titrated on vero e6 cells as described above. essentially the same protocol was used for endogenously produced no [7] : 10 ng/ml il-1b and/or 400 u/ml ifn-c was added to the media 1 h after virus infection. l-nmma (1 mm), a general nos inhibitor, or d-nmma as control for l-nmma, was added to the cells at 1 and 24 h after virus infection. supernatants were collected for virus titration 48 h post infection. an mtt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2h-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 [35] . production of no in vitro, and release of no from snap and of peroxynitrite from sin-1, 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 (1% sulfanilamide and 0.1% naphtylethylenediamide, in 5% phosphoric acid), and the optical density at 540 nm was measured by spectrophotometry. the nitrite standard was diluted in the same medium as used for the samples. vero e6 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 (50 mm tris-hcl, 150 mm nacl, 1 mm edta, 20 mm naf, 100 mm na 3 vo 4 , 1% triton x-100, 1 mm phenylmethylsulfonyl fluoride, 10 lg/ml aprotinin and leupeptin). lysates were mixed 1 : 1 in sample buffer (10 mm tris-hcl, ph 7.5, 0.5% sds, 10% glycerol, 2% b-mercaptoethanol and bromophenol blue) resolved in 10% tris-glycine polyacrylamide gels and transferred to nitrocellulose membranes. blocking was performed overnight at 4 c in 5% nonfat dry milk in 0.1% tween-20 in pbs. the membranes were subsequently incubated with hantavirus n-specific mab 1c12 [36] and b-actin-specific mab for 1 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) 6 . taqman real-time pcr was performed with 300 nm of primer 983f 5 0 -gtgcaccagatcggtgtcc-3 0 , 900 nm of primer 1038r 5 0 -caattcagccatcccagca-3 0 and 200 nm of taqman mgb probe 1003t 5 0 -cctacatgcatttatg-3 0 on a 7900ht sequence detection system (applied biosystems, foster city, ca) with software sds version 2.0 [37] . rna extracted from stocks of puuv kazan-e6 with known concentrations of virus (measured as ffu on vero e6 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 2-deficient mice but not saaremaa, hantavirus is lethal and induces nitric oxide production in suckling mice il-4 and il-10 antagonize il-12-mediated protection against acute vaccinia virus infection with a limited role of ifn-c and nitric oxide synthetase 2 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 c57/bl6 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-349689-njb6619x 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: 2010-03-24 journal: j med virol doi: 10.1002/jmv.21663 sha: doc_id: 349689 cord_uid: njb6619x 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 32 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 5–20 µm. concurrent addition of drug with virus, or treatment of cells prior to infection drastically reduced virus infectivity and viral genome copy number by ≥99.99%. the maximum inhibitory effect of chloroquine was observed within 1–3 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. 82: 817–824, 2010. © 2010 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 2005, chikv infection has assumed epidemic proportions in asia and sub-saharan africa. several outbreaks of chikv fever occurred in 2006, 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 1.4 million cases have been reported [charell et al., 2007; mavalankar et al., 2007; pialoux et al., 2007] . recent cases of chikv infection in europe and italy have occurred as a result of travel to and from infected areas [rezza et al., 2007] . 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 $11.8 kb [jupp and mcintosh, 1998 ]. chikv causes an acute illness characterized by fever, headache, skin rash, vomiting, myalgia, and polyarthralgia [jupp and mcintosh, 1998 ]. 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., 2003 [savarino et al., , 2006 . chloroquine as an effective anti-viral therapeutic for the clinical management of viral diseases was first established in the 1990s for hiv-1 infection [savarino, 2005] . anti-viral effects of chloroquine against sars-cov, hiv type 1 and hepatitis b virus have also been reported [kouroumalis and koskinas, 1986; tsai et al., 1990; vincent et al., 2005] , and the use of chloroquine as a therapeutic for hiv-1 infection is currently being evaluated in clinical trials [savarino et al., 2006] . 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., 2004] . increased virulence of chikv as a result of evolutionary adaptation during chikungunya outbreaks has been reported [schuffenecker et al., 2006; santhosh et al., 2008] . 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 1.1 g sodium bicarbonate/l, 10% heatinactivated fetal bovine serum (fbs), 2 mm l-glutamine, and 80 u of gentamycin. the chivk isolate drde-06, which belongs to the ecs african genotype, was used in the present study . increasing concentrations of chloroquine (5, 10, and 20 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 24 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 1-6 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 25 cm 2 cell culture flask at a density of 1 â 10 5 cells/ml (1 â 10 6 cells per flask) and then incubated for 24 hr. cells were infected with chikv at a multiplicity of infection (m.o.i.) of 0.1. drug was administered to the three different treatment groups (24 hr pre-treatment, concurrent treatment, and post-treatment 1 hpi). infection was allowed to proceed for 36 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 1,100g for 10 min. virus yield was determined by the plaque assay. cells were seeded on 24-well culture plates (greiner bio-one, solingen, germany) at a density of 1 â 10 5 cells/ well and allowed to grow to 95% confluency. the medium was discarded and the cell monolayer was infected with chikv (100 pfu/well). seeded cells were treated with drug 24 hr before infection, concurrent with infection, or 1 hpi. after a period of 1 hr to allow for virus adsorption, cells were overlaid with an overlay medium containing 1.5% methylcellulose, 2% fcs, and the appropriate concentration of drug. after 72 hr, the overlay medium was removed and the infected cell monolayer was fixed in 10% pbs-formaldehyde. virus plaques that formed on vero cells were visualized by staining with 1% 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 36 hpi by neutral red dye uptake assay [finter, 1969] . a selectivity index for each test compound for the pre-treatment, concurrent, and post-treatment (1 hpi) groups was determined as the ratio of the concentration of test compound required to reduce cell viability by 50% (cc 50 ) to the concentration required to inhibit virus infectivity by 50% as compared to control cells (ic 50 ). vero cell monolayers cultured in 25 cm 2 flasks were infected with chikv (m.o.i. of 0.1) to 95% confluence. increasing concentrations of drug (5, 10, and 20 mm) were added to all treatment groups (pre-treatment, concurrent, and post-treatment 1 hpi). infection was allowed to proceed for 36 hr, at which time 1 ml of culture supernatant was drawn from each treatment group in triplicate and then pooled. genomic viral rna was extracted from 140 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 e1 gene was amplified using the following specific primers: 5 0 -acgcaattgagcgaag-cac-3 0 (forward), 5 0 -ctgaagacattggccccac-3 0 (reverse). real-time rt-pcr was performed using the mx 3000p 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 mx3000 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 24-well plates were infected with chikv in duplicate, and then treated with chloroquine at a concentration of 20 mm for increasing periods of time post-infection (1 to 6 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 14 hr. cells were washed five times with pbs, and then fixed using chilled methanol. cells were permeabilized using 0.1% triton-x100 for the detection of intracellular virus. fixed cells were incubated with rabbit anti-chikv hyperimmune serum (1:2,000 dilution) followed by fitc-conjugated anti-rabbit igg (sigma, st. louis, mo) (1:100). cells were washed and then observed using a carl-zeiss aximot 2 (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 0.1 with chikv, and then treated with appropriate concentrations of ammonium chloride, ribavirin and chloroquine 24 hr before and 6 hr after chikv infection. in the case of pre-treatment, compounds were removed by washing before infection. cell viability was measured 36 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 20 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 1 â 10 8 pfu/ml was obtained 36 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 (1 hpi) with chloroquine also inhibited chikv infection at higher concentrations. viral titer was reduced nearly 99% by 20 mm chloroquine, as indicated by the 2-3 log decrease in virus yield in all treatment groups. these results provided substantial evidence of the anti-chikv activity of chloroquine (fig. 1a) . anti-chikv activity was also evaluated by plaque reduction assay. in the presence of 20 mm chloroquine, plaque formation was inhibited 94%, 70%, and 65% in the pre-treatment, concurrent, and post-treatment (1 hpi) groups, respectively (fig. 1b) . 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 450 nm (od 540 ) of treated and untreated cells, ic 50 , ic 90 , and a selectivity index were calculated (table i) . pre-treatment with chloroquine was the most effective anti-chikv strategy, as indicated by a nearly 2.5-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 e1 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 2a -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 2-3 log reduction in viral load in a dose-dependent manner (fig. 2e) . 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. 3) . as compared to infected cells, chloroquine pre-treated infected cells exhibited lower fluorescence intensity, and in the presence of 20 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 3 hpi (fig. 4) . the addition of chloroquine during the early stages of viral infection (1-3 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 6 hpi, based on cell viability (fig. 5a) . 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. 5b) . 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 6 hpi. (fig. 5c ). 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 90% (based on the plaque forming activity of 100 pfu of virus) (fig. 1b) . there was also a significant reduction in viral rna copy number, based on real-time rt-pcr analysis (fig. 2) , 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., 2006] . the data obtained from the current study indicate j. med. virol. doi 10.1002/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 5, 10, and 20 mm chloroquine. the specificity of the amplified products was analyzed by t m curve analysis (a). amplification plots for cells treated with chloroquine 24 hr before (b), concurrently (c), and 1 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., 1984] . 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 1 hpi to significantly inhibit virus yield. the addition of chloroquine at 6 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., 2005] . 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, 1998 ]. it has been reported that a low endosomal ph is also required for chikv entry into cells [sourisseau et al., 2007] . in the case of concurrent treatment and post-treatment (1 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 3 hpi (fig. 4) . in the post-treatment group, the addition of chloroquine at an early stage (1-3 hpi) of infection had a marked effect on virus yield, whereas late stage addition (4-6 hpi) was ineffective. the ic 50 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, 1990] . 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. 3) . the results of ift also supported the finding that pre-treatment of cells with 10 or 20 mm chloroquine was more effective than concurrent treatment and posttreatment (1 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, 1986] . 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., 1984] . ammonium chloride was effective against chikv when cells were pre-treated (24 hr before), and retained its anti-viral activity even when it was removed prior to infection. however, administration of ammonium chloride 6 hpi did not protect cells from chikv infection (fig. 5a) . 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. 5b) . thus, chloroquine (fig. 5c) 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-21 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 a226v shift in 2007 indian chikungunya virus isolates expanding the frontiers of existing antiviral drugs: possible effects of hiv-1 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-300379-db79kb5c 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: 2019-04-12 journal: front microbiol doi: 10.3389/fmicb.2019.00718 sha: doc_id: 300379 cord_uid: db79kb5c 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(50)) of 13.87 ± 1.09 μm and 33.33 ± 1.13 μm in vero and a549 cells, respectively; without showing any cytotoxic effect in both cell lines (median cytotoxic concentration (cc(50)) > 1,000 μm). moreover, ata protected both cell types from zikv-induced cytopathic effect (cpe) and apoptosis in a timeand concentration-dependent manner. in addition, pre-treatment of vero cells with ata for up to 72 h also resulted in effective suppression of zikv replication with similar ic(50). 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 ∼10.7 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 (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5) proteins avila-perez et al., 2018) . zika virus was initially isolated from uganda in 1947 and viral infections only occurred sporadically in africa and asia until 2007. zikv appeared explosively as the first large-scale outbreak occurred in the yap island in 2007 and french polynesia in 2013 (weaver et al., 2016) . most recently, in 2015, the first local transmission of zikv was found in territories of latin america and the caribbean, resulting in up to 1.3 million of zikv infection suspected cases (tang et al., 2016; tripathi et al., 2017) . 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., 2016; fink et al., 2018) . upon infection, zikv can be shed in blood, urine, semen, saliva, amniotic fluid, breast milk, and cerebrospinal fluid (nayak et al., 2016; colt et al., 2017; nazerai et al., 2019) . most people (75∼80%) 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., 2018) . in rare cases, people with symptoms may have neurological guillain-barré syndrome complications (oehler et al., 2014; rivera-concepcion et al., 2018; nazerai et al., 2019) . 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 2015 (lessler et al., 2016) . 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 2016 (lazear and diamond, 2016; ramos da silva and gao, 2016; weaver et al., 2016; tripathi et al., 2017) . there are several vaccines and antiviral drugs currently under development for the prevention or treatment of zikv infection larocca et al., 2016; shan et al., 2017; fink et al., 2018) . dna-based larocca et al., 2016) , inactivated larocca et al., 2016; shan et al., 2017) , live-attenuated and mrna (richner et al., 2017) vaccines have been proposed for the prophylactic treatment of zikv infections. on the other hand, arbidol (arb) (fink et al., 2018; haviernik et al., 2018) , bortezomib, mycophenolic acid, daptomycin (barrows et al., 2016) , obatoclax, saliphenylhalamide, gemcitabine (kuivanen et al., 2017) , emetine (yang et al., 2018) , and sofosbuvir (bullard-feibelman et al., 2017) 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 2 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., 2017) . ade, which has been extensively described in denv (priyamvada et al., 2017) , 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., 2017) . because of the structural similarities between denv and zikv, denv immunity-linked ade of zikv infection has also been reported priyamvada et al., 2017) . 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., 2003) , cryptosporidium parvum (klein et al., 2008) , human immunodeficient virus (hiv) (mitra et al., 1996; de clercq, 2005) , hepatitis c virus (hcv) mukherjee et al., 2012; shadrick et al., 2013) , vaccinia virus (myskiw et al., 2007) , influenza virus (hung et al., 2009) , enterovirus 71 (hung et al., 2010) and severe acute respiratory syndrome coronaviruses (sars-cov) (he et al., 2004) . mechanistic studies have suggested that ata has the ability to modulate various cellular enzymes such as activators of the janus kinase 2 (jak2) and signal transducer and activator of transcription 5 (stat5) families (rui et al., 1998) , inhibitors of nucleases (shadrick et al., 2013) , glucose-6-phosphate dehydrogenase (bina-stein and tritton, 1976) , and topoisomerase ii proteins (catchpoole and stewart, 1994; benchokroun et al., 1995) as well as the enzymatic activity of the vaccinia virus ah1l phosphatase (smee et al., 2010) . 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 1,000 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-81) and human adenocarcinoma alveolar basal epithelial a549 (atcc ccl-185) cells were maintained in dulbecco's modified eagle's medium (dmem; mediatech, inc.) supplemented with 5% fetal bovine serum (fbs) and 1% psg (100 u/ml penicillin, 100 µg/ml streptomycin, and 2 mm l-glutamine) at 37 • c in a 5% co 2 atmosphere. paraiba/2015 zikv isolate was kindly provided by stephen dewhurst (department of microbiology and immunology, university of rochester). uganda/1947 (mr_766 strain, catalog no. nr-50065) and nigeria/1968 (ibh 30656 strain, catalog no. nr-50066) zikv isolates were obtained from the biodefense and emerging infections research resources repository (bei resources). puerto rico/2015 (prvabc59 strain) and french polynesia/2013 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., 2018) . aurintricarboxylic acid (catalog no. a1895) and arbidol (arb, catalog no. slm0860) were purchased from sigma-aldrich, mo, united states. both compounds were prepared at 100 mm stock solution dissolved in dimethyl sulfoxide (dmso) and kept at −20 • c until experimental use. each drug was diluted into infectious media (dmem 2% fbs, 1% psg) for the described experiments, where the maximum dmso concentration was 0.1%. cell viability in vero and a549 cells was measured using the celltiter 96 non-radioactive cell proliferation assay (promega) following the manufacturer's instructions. briefly, confluent vero or a549 cells (96-well plate format, 5 × 10 4 cells/well, triplicates) were treated with 100 µl of dmem containing serially diluted (twofold dilutions, starting concentration of 1,000 µm) chemicals or 0.1% dmso (vehicle control). plates were incubated at 37 • c in a 5% co 2 atmosphere for 36 or 72 h. samples were treated with 15 µl of dye solution and incubated at 37 • c in a 5% co 2 atmosphere for 4 h. next, cells were treated with 100 µl of solubilization solution/stop mix and absorbance at 570 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 50 ) were calculated using graphpad prism software version 8.0. confluent monolayers (96-plate format, 5 × 10 4 cells/well, triplicates) of vero cells were infected with 25 plaque forming units (pfu)/well of paraiba/2015 , uganda/1947 , nigeria/1968 , puerto rico/2015 , and french polynesia/2013 at 37 • c in infection media. after 1 h of adsorption, virus inoculum was removed and cells were washed three times with infection media before adding fresh infection media containing 1% microcrystalline cellulose (avicel, sigma-aldrich) and the indicated concentration of compounds, or 0.1% dmso as vehicle control. in case of pre-treatment experiments, the cell monolayers were treated with the indicated concentration of compound, or 0.1% dmso, for the indicated times before zikv infection. infected cells were incubated at 37 • c for 36-60 h, depending on virus strains. for immunostaining, cells were fixed with 4% paraformaldehyde for 1 h, washed three times with phosphate buffered saline (pbs) and permeabilized with 0.2% triton x-100 for 10 min at room temperature. then, the plates were blocked with 1.25% bovine serum albumin (bsa) in pbs (blocking solution) for 1 h at room temperature, followed by incubation with 1 µg/ml of the pan-flavivirus envelop (e) protein monoclonal antibody 4g2 (atcc, catalog no. vr-1852) diluted in blocking solution for 1 h at 37 • 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., 2015) . non-linear regression curves and the median inhibitory concentration (ic 50 ) were determined as described above. confluent monolayers (24-well plate format, 2.5 × 10 5 cells/well, triplicates) of vero or a549 cells were infected (multiplicity of infection, moi, 0.1) with paraiba/2015 diluted in infection media for 1 h at room temperature. after viral absorption, cells were incubated with infection media containing the indicated concentrations (250, 25, 2.5, and 0 µm) of ata. at 12, 24, 48, and 72 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., 2018) . levels of apoptosis were measured using the caspase-glo r 3/7 assay (promega, wi, united states) following the manufacturer's instruction. briefly, vero and a549 cells (24-well plate format, 2.5 × 10 5 cells/well, triplicates) were infected with zikv paraiba/2015 (moi of 0.1) and, at the indicated times post-infection, cells and tissue culture supernatants were collected and centrifuged. twenty five microliters of supernatants were mixed with 25 µl of caspase-3/7 reagent using a plate shaker, incubated at room temperature for 1 h, and luminescence at 570 nm was measured using a spectramax id5 (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 < 0.0332, * * p < 0.0021, * * * p < 0.0002, * * * * p < 0.0001. all data were analyzed with prism software version 8.00 (graphpad software, ca, united states). cc 50 and ic 50 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 50 with the ic 50 . before examining the inhibitory effect of ata (figure 1 ) against zikv infection, we first determined the cc 50 of ata on vero and a549 cells (figure 2) . for this, we treated both cell lines with serial (twofold) dilutions of ata and measured cell viability at 36 and 72 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., 2018) and a549 (fink et al., 2018) cells. we did not observe any toxicity with ata in vero (figure 2a ) or a549 ( figure 2b ) cells at 36 or 72 h post-treatment, even at the highest concentration tested (1,000 µm), while arb showed cc 50 values of 74.71 ± 1.09 or 59.37 ± 1.10 µm in vero ( figure 2c ) and 114.6 ± 1.08 or 91.0 ± 1.08 µm in a549 ( figure 2d ) cells (table 1) at 36 or 72 h post-treatment, respectively. to determine the ic 50 of ata, vero and a549 cells were infected with 25 pfu/well of paraiba/2015 and after 1 h of viral absorption, virus inoculum was replaced with infection media with twofold serial dilutions (starting concentration of 1,000 µm) of ata or arb (figure 3 ) and the ic 50 calculated as described in the section "materials and methods." although the ic 50 of ata ( figure 3a) and arb ( figure 3c) in vero cells were similar (13.87 ± 1.09 µm and 18.19 ± 1.6 µm, respectively), the selective index (si, cc 50 /ic 50 ) of ata (>72.10) was significantly higher than that of arb (4.11) ( table 1) . likewise, the ic 50 of ata ( figure 3b ) and arb ( figure 3d ) in a549 cells were similar but with clearly different si values (>26.26 for ata and 2.21 for arb) ( table 1) . notably the cc 50 , ic 50 , and si of arb were similar to those previously described in the literature in these cell lines (fink et al., 2018; haviernik et al., 2018) . 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 250 µm of ata in vero ( figure 4a ) and a549 ( figure 4b ) cells while 2.5 µm and 25 µm concentrations of ata showed partial viral inhibition in vero and a549 cells (figures 4a,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 5) . to that end, vero and a549 cells were infected (moi 0.1) with paraiba/2015 and, after 1 h of viral absorption, cells were treated with 0, 2.5, 25, and 250 µm of ata. at 48 h p.i., cells were observed under a light microscope for evaluation of their morphology and cpe ( figure 5a ). as expected from our previous results, 25 µm and more clearly 250 µm of ata were able to prevent zikv-induced cpe in both cell lines (figure 5a) . to quantify the ability of ata to prevent zikv-induced apoptosis, tissue culture supernatants from zikv-infected vero and a549 cells were harvested at 24, 48, and 72 h p.i. to measure the level of apoptotic signal as determined by caspase 3 and 7 activities ( figure 5b) . zikv-infected cells showed figure 4 | aurintricarboxylic acid inhibition of zikv replication: vero (a) and a549 (b) cells (24-well plate format, 2.5 × 10 5 cells/well, triplicates) were infected (moi 0.1) with paraiba/2015. tissue culture supernatants were collected at 24, 48, and 72 h p.i., and viral titer were calculated by immunostaining (fluorescent forming units, ffu/ml). dotted line indicates the limit of detection (20 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 < 0.0332, * * p < 0.0021, * * * p < 0.0002, * * * * p < 0.0001, or no significance (n.s.). figure 5 | aurintricarboxylic acid protects vero and a549 cells from zikv-induced cell death: vero and a549 cells (24-well plate format, 2.5 × 10 5 cells/well, triplicates) were infected (moi 0.1) with paraiba/2015. after 1 h viral adsorption, cells were treated with the indicated concentrations (250, 25, 2.5, and 0 µm) of ata. at 48 h p.i., cells were observed and imaged under an optical microscope. scale bar = 100 µm. (a) caspase 3/7 levels were measured in the tissue culture supernatants at 24, 48, and 72 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 < 0.0332, * * p < 0.0021, * * * p < 0.0002, * * * * p < 0.0001, or no significance (n.s.). increased caspase 3 and 7 levels up to eightfolds in vero cells and up to 4.2-folds in a549 cells compared to mock-infected cells ( figure 5b ). levels of caspase 3 and 7 activation were dose-dependently reduced by ata with 250 µm of ata showed only 0.4-and 1.7-fold induction as compared to mock-infected vero and a549 cells, respectively ( figure 5b ). we next determined whether ata is able to inhibit both ancestor african (uganda/1947 and nigeria/1968 ) and contemporary asian/american (puerto rico/2015 and french polynesia/2013) zikv lineage strains using our microplaque reduction assay (figure 6) . we observed similar ic 50 values of ata with uganda/1947 ( figure 6a , ic 50 = 15.07 ± 1.19 µm), nigeria/1968 ( figure 6b , ic 50 = 15.97 ± 1.02 µm), puerto rico/2015 ( figure 6c , ic 50 = 17.55 ± 1.16 µm), and french polynesia/2013 ( figure 6d , ic 50 = 13.92 ± 1.01 µm), compared to those observed with paraiba/2015 (figure 3 and table 1 ), 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 7) . to that end, vero cells were pre-treated with ata for 12 ( figure 7a) , 24 ( figure 7b) , 48 (figure 7c) , or 72 ( figure 7d ) h prior to infection (moi 0.1) with paraiba/2015. pre-treatment with ata for 12-72 h before zikv infection resulted in similar ic 50 values (14.33 ± 1.06 µm, 13.18 ± 1.05 µm, 12.59 ± 1.02 µm, and 10.50 ± 1.05 µm; respectively) demonstrating that ata is stable and able to prevent zikv infection even when administered 3 days previous to viral infection (figure 7 and table 1 ). 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., 2016; lessler et al., 2016; shan et al., 2017; fink et al., 2018) . 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 1 ) has limited toxicity (figure 2) and an effective and dose-dependent antiviral activity against zikv infection (figures 3, 4) in both monkey kidney epithelial vero and human alveolar a549 cells. notably, ata can prevent zikv-induced cpe and apoptosis in both cell lines ( figure 5 ) and has broad anti-viral activity against representative zikv strains from the african (uganda/1947 and nigeria/1968) and the asian/american (puerto rico/2015 and french polynesia/2013) lineages (figure 6) . moreover, ata can also prevent zikv infection even when administered 3 days before infection (figure 7) . aurintricarboxylic acid is a polyanionic aromatic compound that structurally relates to suramin (balzarini et al., 1986) and is believed to influence over 100 host and viral enzymes (shadrick et al., 2013) . 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 ns3 helicase and prevent its binding to either atp or nucleic acids, as previously described for hcv (mukherjee et al., 2012; shadrick et al., 2013) . likewise, ata could inhibit the zikv rna-dependent rna polymerase (rdrp) ns5 protein, as described for hcv mukherjee et al., 2012; shadrick et al., 2013) and enterovirus 71 (hung et al., 2010) . similarly, ata could inhibit the methyltransferase activity of ns5 involved in mrna capping processes, as previously described for other flaviviruses (denv and yfv) (milani et al., 2009; garcia et al., 2017) . because of the structural similarities between denv and zikv ns5 proteins, it is feasible that, similar to denv, ata binds to ns5 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., 2002) . it is possible that the anti-apoptotic activity of ata protects against zikv-induced cell death, as demonstrated in this study (figure 5) . notably, it has been recently shown that zikv infection induced apoptosis through caspase 3 and 9 in a549 cells and through caspase 3 in neonatal mice brain (huang et al., 2016; frumence et al., 2016) . 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 2015 to february 2016, a total of 116 residents from 33 states in the united states were diagnosed with zikv infection (armstrong et al., 2016) . out of 115 patients, 110 (96%) traveled to areas of active zikv transmission before the infection and five (4%) did not travel but reported sexual contact with a traveler who had a symptomatic illness (armstrong et al., 2016) . 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 72 h prior to infection with zikv showed similar ic 50 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., 2003; he et al., 2004; de clercq, 2005; myskiw et al., 2007; klein et al., 2008; chen et al., 2009; hung et al., 2009 hung et al., , 2010 mukherjee et al., 2012; shadrick et al., 2013) that are present in zikv endemic areas, treatment with ata could be used for the broad prevention of denv, yfv (milani et al., 2009; shadrick et al., 2013; garcia et al., 2017) , hcv mukherjee et al., 2012; shadrick et al., 2013) , and parasitic infestation (cryptosporidium parvum) (klein et al., 2008) 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., 1990) , apoptosis (roberts-lewis et al., 1993; heiduschka and thanos, 2000) , parasite infestations (klein et al., 2008) , bacterial (y. pestis) (liang et al., 2003) , and vaccinia virus (smee et al., 2010) infections. in the case of vaccinia virus, ata did not protect mice from a lethal challenge at a dose of 30 mg/kg/day (smee et al., 2010) . 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 a549 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 = 72.1 in vero cells and 26.26 in a549 cells) as compared to other previously described drugs, including emetine (si = 9.84 in snb-19 cells and 2.88 in env+ cells) (yang et al., 2018) , obatoclax [si = 65 in human retinal pigment epithelial (rpe) cells] (kuivanen et al., 2017) , saliphenylhalamide (si > 200 in rpe cells) (kuivanen et al., 2017) , gemcitabine (si > 1,000 in rpe cells) (kuivanen et al., 2017) , sofosbuvir (si > 52.63 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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 jak2-stat5 signaling pathway in nb2 lymphoma cells by an anti-apoptotic agent, aurintricarboxylic acid aurintricarboxylic acid modulates the affinity of hepatitis c virus ns3 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-331094-22366b81 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-2 infection date: 2020-06-13 journal: viruses doi: 10.3390/v12060642 sha: doc_id: 331094 cord_uid: 22366b81 as of june 2020, the number of people infected with severe acute respiratory coronavirus 2 (sars-cov-2) continues to skyrocket, with more than 6.7 million cases worldwide. both the world health organization (who) and united nations (un) has highlighted the need for better control of sars-cov-2 infections. however, developing novel virus-specific vaccines, monoclonal antibodies and antiviral drugs against sars-cov-2 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-2 strain and vero-e6 cells. we identified the most potent sera from recovered patients for the treatment of sars-cov-2-infected patients. we also screened 136 safe-in-man broad-spectrum antivirals against the sars-cov-2 infection in vero-e6 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-19. every year, emerging and re-emerging viruses such as sars-cov-2, 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 [1, 2] . as of june 2020, the number of people infected with sars-cov-2 continues to rise, with more than 6.7 million cases worldwide. we selected subjects among healthy individuals, in-and outpatients, as well as patients recovered from sars-cov-2 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-19 is suspected" (who/2019-ncov/clinical/2020.4). 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: nct04320732; rek: 124170). human telomerase reverse transcriptase-immortalized retinal pigment epithelial (rpe), lung adenocarcinoma a427, non-small-cell lung cancer calu-3 and epithelial colorectal adenocarcinoma caco-2 cells were grown in dmem-f12 supplemented with 100 µg/ml streptomycin and 100 u/ml penicillin (pen-strep), 2 mm l-glutamine, 10% fbs and 0.25% sodium bicarbonate (sigma-aldrich, st. louis, mo, usa). human neural progenitor cells derived from ips cells were generated and maintained as described previously [20] . human large-cell lung carcinoma nci-h460, colon cancer sw620, colorectal carcinoma sw480 and ht29 cells were grown in rpmi medium supplied with 10% fbs and pen-strep. human adenocarcinoma alveolar basal epithelial a549, human embryonic kidney hek-293 cells and kidney epithelial cells extracted from an african green monkey (vero-e6) were grown in dmem supplemented with 10% fbs and pen-strep. the cell lines were maintained at 37 • c with 5% co 2 . the sars-cov-2 strains were isolated and propagated in a biological safety level 3 (bsl-3) facility. two hundred microliters of nasopharyngeal swabs (nps) in universal transport medium were diluted 5 times in culture medium (dmem) supplemented with 0.2% bovine serum albumin (bsa), 0.6 µg/ml penicillin, 60 µg/ml streptomycin and 2 mm l-glutamine and inoculated into vero-e6 cells. after 4 days of incubation, the media were collected, and the viruses were passaged once again in vero-e6 cells. after 3 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_v2 protocol, a modified version of the bomb-protocol [21] . the eluate (2.5 or 5 µl) was analyzed by rt-qpcr using a cfx96 real-time thermal cycler (bio-rad, hercules, ca, usa) as described elsewhere [22] , with some modifications. one 20-µl reaction contained 10 µl of qscript xlt one-step rt-qpcr toughmix (2×) (quanta biosciences, beverly, ma, usa), 1 µl each of the primer and probe with final respective concentrations of 0.6 and 0.25 µm and 2 µl of molecular-grade water. thermal cycling was performed at 50 • c for 10 min for reverse transcription, followed by 95 • c for 1 min and then 40 cycles of 95 • c for 3 s and 60 • c for 30 s. for testing the production of infectious virions, we titered the viruses as described in our previous studies [23] [24] [25] . in summary, media from the viral culture were serially diluted from 10 −2 to 10 −7 in serum-free dmem containing 0.2% bovine serum albumin. the dilutions were applied to a monolayer of vero-e6 cells in 6 or 24-well plates. after one hour, cells were overlaid with virus growth medium containing 1% carboxymethyl cellulose and incubated for 96 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_v2 protocol, a modified version of the bomb-protocol. libraries were prepared using a nugen trio rna-seq kit. sequencing was performed on the nextseq500 instrument (ns500528; setup: pe 2 × 75 bp + single index (8 bp)) using a nextseq mid150 sequencing kit and nextseq mid flow cell (ncs version: 2.2.0.4). reads were aligned using the bowtie 2 software package version 2.3.4.1 to the reference viral genome wuhan-hu-1/2019. sequence alignments were converted to binary alignments and sorted using samtools version 1.5. the consensus fastq sequences were obtained with bcftools and vcfutils.pl (from samtools) and converted to fasta using seqtk (https://github.com/lh3/seqtk). viral genomes in fasta formats were submitted to www.gisaid.org. the accession numbers of these genomes are: epi_isl_450352 (hcov-19/norway/trondheim-e10/2020), epi_isl_450351 (hcov-19/norway/trondheim-e9/2020), epi_isl_450350 (hcov-19/norway/trondheim-s15/2020), epi_isl_450349 (hcov-19/norway/trondheim-s12/2020), epi_isl_450348 (hcov-19/norway/ trondheim-s10/2020), epi_isl_450347 (hcov-19/norway/trondheim-s5/2020) and epi_isl_450346 (hcov-19/norway/trondheim-s4/2020). transmission of the strains was visualized using https: //nextstrain.org/ncov/global?f_author=aleksandr%20ianevski%20et%20al. the virus (multiplicity of infection, moi 0.1) was aliquoted in eppendorf tubes and incubated at −80, −20, 4, 20, 37 and 50 • c for 48 h or at 96 • c for 10 min. alternatively, the virus was aliquoted in wells of a 96-well plate without a lid. the virus was exposed to uvc light (λ = 254 nm, ≥125 µw/cm 2 ) for 10, 20, 40, 80, 160, 320 and 640 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-e6 cells were infected with the virus for 72 h, and cell viability was measured using a celltiter-glo assay (promega, madison, wi, usa). luminescence was read using a plate reader. approximately 4 × 10 4 vero-e6 cells were seeded per well in 96-well plates. the cells were grown for 24 h in dmem supplemented with 10% fbs and pen-strep. serum samples were diluted 100-fold, and protein concentrations were quantified using nanodrop. serum samples were prepared in 3-fold dilutions at 7 different concentrations, starting from 1 mg/ml in the virus growth medium (vgm) containing 0.2% bsa and pen-strep in dmem. virus hcov-19/norway/trondheim-e9/2020 was added to the samples to achieve a moi of 0.1 and incubated for 1h at 37 • c. 0.1% dmso was added to the control wells. the vero-e6 cells were incubated for 72 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 [26] . supplementary materials table s1 lists these and other potential bsaas, their suppliers and catalogue numbers. the control wells. the cells were mock-or hcov-19/norway/trondheim-e9/2020-infected at a moi of 0.1. after 72 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 50 ) 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 7.0a (graphpad software, san diego, ca, usa). the half-maximal effective concentrations (ec 50 ) were calculated based on the analysis of the viability of infected cells by fitting drug dose-response curves using the four-parameter (4pl) 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 50 or cc 50 ) and λ is the steepness (slope) of the curve. a relative effectiveness of the drug was defined as the selectivity index (si = cc 50 /ec 50 ). the threshold of the si used to differentiate between active and inactive compounds was set to 3. 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 10%. vero-e6 cells were treated with different concentrations of a combination of two bsaas. after 72 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 2 [27] . 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 1.0 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-e6 cells were treated with nelfinavir, amodiaquine or both drugs at indicated concentrations. cells were infected with the hcov-19/norway/trondheim-e9/2020 strain at moi 0.1 or mock. after 24 h, total rna was isolated using rneasy plus mini kit (qiagen, hilden, germany). libraries were prepared and sequenced on a nextseq500 (ns500528) instrument (set up: pe 2 × 75 bp + single index 8 bp) using a nextseq mid150 sequencing kit, nextseq mid flow cell, ncs version: 2.2.0.4. reads were aligned using the bowtie 2 software package version 2.3.4.1 to the ncbi reference sequence for sars-cov-2 (nc_045512.2) and to the human grch38 genome. number of mapped and unmapped reads that aligned to each gene were obtained with the featurecounts function from rsubread r-package version 2.10. the gff table for the reference sequence was downloaded from https://ftp.ncbi.nlm.nih.gov/genomes/all/gcf/009/858/895/gcf_ 009858895.2_asm985889v3/gcf_009858895.2_asm985889v3_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 log2-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-2 pandemic and have summarized the information in a database that can be freely accessed at https://sars-coronavirus-2.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 [28] [29] [30] , as well as other public sources. the website was developed with php v7 technology using d3.js v5 (https://d3js.org/) for visualization. the covid-19 statistics are automatically exported from the covid-19 data repository by the center for systems science and engineering (csse) at johns hopkins university (https://github.com/cssegisanddata/covid-19). we isolated seven sars-cov-2 strains from 22 nps samples of covid-19 patients using vero-e6 cells. the rt-qpcr cycle threshold (ct) values were 18-20 before and 13-15 after propagation of the viruses in vero-e6 cells (figure 1a ,b). we sequenced seven strains and found that the sequences differed from the reference hcov-19/wuhan/wiv04/2019 strain by a few missense mutations ( figure 1c) . phylogenetic analysis showed a close relationship between the strains (figure 1d ). in cross-referencing our sequence data with the pathogen-tracking resource nextstrain.org, we determined that the sars-cov-2 strains isolated in trondheim originated from china, denmark, the usa and canada (figure 1e) . in addition, we tested several cell lines and found that vero-e6 was the most susceptible for virus-mediated death and virus amplification (figure s1a-c). to establish the rationale for safe work, we incubated the virus at different temperatures for 48 h or exposed to uvc radiation for different time periods. the resulting virus preparations were analyzed by rt-qpcr and used to infect vero-e6 cells. virus incubation at 37 • c for 48 h or uvc exposure for 10 sec destabilized the virus and rescued infected cells from virus-mediated death ( figure s2 ). we evaluated the neutralization capacity of seven serum samples obtained from patients recovered from covid-19. 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-19 had serum sensitivity scores (sss) > 5 and rescued >50% cells from virus-mediated death at 1 mg/ml (figure 2a,b) . notably, that serum from a recovered patient with sss = 7.2 neutralized all seven sars-cov-2 strains ( figure s3 ). our neutralization test of 32 samples (table s2) showed a moderate positive correlation with igg (r = 0.59, p < 0.001) and igm (r = 0.43, p = 0.01) s/co values obtained using commercial elisa kits that recognize the sars-cov-2 n protein (figure 2c,d) . however, the correlation between the igg and igm elisa results was only r = 0.28, p = 0.11. furthermore, we found a moderate negative correlation between ssss and time intervals between the sars-cov-2 diagnosis and serum collection for 66 samples (-0.5, p < 0.025; figure 2d ). altogether, these results suggest that patients diagnosed with covid-19 produce different immune responses to the sars-cov-2 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-19. 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-19 had serum sensitivity scores (sss) > 5 and rescued >50% cells from virus-mediated death at 1 mg/ml (figure 2a,b) . notably, that serum from a recovered patient with sss = 7.2 neutralized all seven sars-cov-2 strains ( figure s3 ). our neutralization test of 32 samples (table s2) showed a moderate positive correlation with igg (r = 0.59, p < 0.001) and igm (r = 0.43, p = 0.01) s/co values obtained using commercial elisa kits that recognize the sars-cov-2 n protein (figure 2c,d) . however, the correlation between the igg and igm elisa results was only r = 0.28, p = 0.11. furthermore, we found a moderate negative correlation between ssss and time intervals between the sars-cov-2 diagnosis and serum collection for 66 samples (−0.5, p < 0.025; figure 2d ). altogether, these results suggest that patients diagnosed with covid-19 produce different immune responses to the sars-cov-2 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 [17] [18] [19] 26, 31] . some of these agents could be repositioned for the treatment of a sars-cov-2 infection. we tested 136 agents against sars-cov-2 in vero-e6 cells. remdesivir was included as a positive control [32] and nicotine as a negative control. seven different concentrations of the compounds were added to virus-infected cells. cell viability was measured after 72 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 285 to 585; table s1 ). the compounds we identified possessed a structure-activity relationship (figure 3a) . auc for remdesivir was 290. interestingly, 10 µm of nicotine rescued cells from virus-mediated death but altered the cell morphology (auc = 239; figure s4 ). 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 3b ,c). importantly, amodiaquine had a superior si over its analogs chloroquine, hydroxychloroquine, quinacrine and mefloquine ( figure s5 ). thus, we identified and validated anti-sars-cov-2 activities for six bsaas in vero-e6 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 4a) . the observed drug combination responses were compared with the expected combination responses calculated by means of the zero-interaction potency (zip) model [27, 33] . we quantified synergy scores, which represent the average excess response due to drug interactions (i.e., 10% of cell survival beyond the expected additivity between single drugs has a synergy score of 10). 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 >10; figure 4b ). moreover, the nelfinavir-amodiaquine treatment was effective against all seven sars-cov-2 strains (figure 4c ). thus, we identified synergistic drug combinations against sars-cov-2 infections. we next profiled transcriptional responses to nelfinavir, amodiaquine or both drugs in virus-or mock-infected vero-e6 cells at 24 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 s6a ). these genes (cxcl1, cxcl2, cxcl3, cxcl8, cxcl10, cxcl11, oasl, ifnl1, mx1 and herc5) 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 s6b) . (a) structure-antiviral activity relation of 136 broad-spectrum antivirals (bsaas). the compounds were clustered based on their structural similarity calculated by ecpf4 fingerprints and visualized using the d3 javascript library. the anti-sars-cov-2 activity of the compounds was quantified using the auc and shown as bubbles. bubble size corresponds to compounds aucs. (b) vero-e6 cells were treated with increasing concentrations of a compound and infected with the hcov-19/norway/trondheim-e9/2020 strain (moi, 0.1) or mock. after 72 h, the viability of the cells was determined using the celltiter-glo assay. mean ± sd; n = 3. (c) table showing half-maximal cytotoxic concentration (cc 50 ), the half-maximal effective concentration (ec 50 ) and selectivity indexes (si = cc 50 /ec 50 ) for selected anti-sars-cov-2 compounds calculated from ctg and plaque assays. mean ± sd; n = 3. figure 3 . anti-sars-cov-2 activity of safe-in man broad-spectrum antivirals in vero-e6 cells. (a) structure-antiviral activity relation of 136 broad-spectrum antivirals (bsaas). the compounds were clustered based on their structural similarity calculated by ecpf4 fingerprints and visualized using the d3 javascript library. the anti-sars-cov-2 activity of the compounds was quantified using the auc and shown as bubbles. bubble size corresponds to compounds aucs. (b) vero-e6 cells were treated with increasing concentrations of a compound and infected with the hcov-19/norway/trondheim-e9/2020 strain (moi, 0.1) or mock. after 72 h, the viability of the cells was determined using the celltiter-glo assay. mean ± sd; n = 3. (c) table showing half-maximal cytotoxic concentration (cc50), the half-maximal effective concentration (ec50) and selectivity indexes (si = cc50/ec50) for selected anti-sars-cov-2 compounds calculated from ctg and plaque assays. mean ± sd; n = 3. to rapidly respond to the covid-19 outbreak, we developed a freely accessible website summarizing novel anti-sars-cov-2 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 542 in-progress and completed clinical trials that test the efficacy of therapeutic agents to treat covid-19 or complications that arise from covid-19. these trials include over 192 unique therapeutic agents in varying combinations and applications. importantly, we list 71 clinical trials that are already completed or are projected to be completed by the end of june 2020. of note, among these are trials of remdesivir, favipiravir, lopinavir/ritonavir, hydroxychloroquine, to rapidly respond to the covid-19 outbreak, we developed a freely accessible website summarizing novel anti-sars-cov-2 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 542 in-progress and completed clinical trials that test the efficacy of therapeutic agents to treat covid-19 or complications that arise from covid-19. these trials include over 192 unique therapeutic agents in varying combinations and applications. importantly, we list 71 clinical trials that are already completed or are projected to be completed by the end of june 2020. of note, among these are trials of remdesivir, favipiravir, lopinavir/ritonavir, hydroxychloroquine, dipyridamole and interferons alpha and beta, which are all phase 3 or 4 clinical trials scheduled to be currently completed. the "prevention" section summarizes 23 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 377 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-2, 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-2 options emerge, or the statuses of existing ones are updated. here, we reported the isolation of seven sars-cov-2 strains from samples of patients suffering from covid-19. full-genome sequencing revealed that the strains were highly similar (98.8%) to one another and to the strains circulating in china, denmark, the usa and canada. all seven strains contain d614g in the s protein. strains with this mutation began spreading in europe in early february and became dominant in other regions (https://doi.org/10.1101/2020.04.29.069054). we screened 12 cell lines for their susceptibility for the sars-cov-2 infection and virus replication. vero-e6 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 [34] . moreover, this cell line was used routinely in anti-sars-cov-2 research [35] [36] [37] [38] . we also showed that >10 sec of uvc radiation or 48 h incubation at 37 • c neutralized sars-cov-2, 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-2 and other viruses [39] [40] [41] [42] . neutralization tests are crucial tools for the assessment of previous sars-cov-2 exposure and potential immunity [12, 13] . we have developed a test to assess the neutralization capacity of serum samples from patients recovered from sars-cov-2 infections, patients with endemic coronavirus infections and healthy blood donors. our results suggest that covid-19 patients respond differently to the sars-cov-2 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-19 for the treatment of sars-cov-2-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 [43] . however, the recent safety study on 5000 hospitalized patients transfused under the u.s. food and drug administration's national expand access program for covid-19 revealed no toxicity (https://doi.org/10.1101/2020.05. 12.20099879) . 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 [44, 45] . 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 0, i and iia). therefore, drug repositioning for covid-19 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 [26, 46, 47] . we tested 136 safe-in-man bsaas against sars-cov-2 in cell cultures. we identified salinomycin, obatoclax, amodiaquine, nelfinavir, emetine and homoharringtonine as having anti-sars-cov-2 activity, which we put forward as potential anti-sars-cov-2 drug candidates. nelfinavir (viracept) is an orally bioavailable inhibitor of human immunodeficiency virus hiv-1 (750 mg per os (po) q8hr). it targets hiv protease for the treatment of hiv infections [48] . molecular docking studies predict that nelfinavir binds to the sars-cov-2 protease [49] . nelfinavir could also inhibit cell fusion caused by the sars-cov-2 s glycoprotein (https://doi.org/10.1101/2020.04. 06.026476) [50] . it also inhibits chikungunya virus (chikv), dengue virus (denv), hepatitis c virus (hcv), herpes simplex virus 1 (hsv-1) and sars-cov infections (https://doi.org/10.1039/ c5ra14469h) [51] [52] [53] . amodiaquine is a medication used to treat malaria. the recommended dose for a course of amodiaquine is 30 mg amodiaquine base/kg body weight over three days, i.e., 10 mg/kg/day [54] . [55] [56] [57] [58] [59] [60] . 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 24 h infusion of obatoclax 25-60 mg/day for three days in two-week cycles or 3 h infusions in a 3-day cycle have previously been evaluated in cancer patients [61] . in addition, obatoclax showed antiviral activity against fluav, zikv, wnv, yfv, sinv, junín virus (junv), lasv, herpes simplex virus 2 (hsv-2), echovirus 1 (ev1), human metapneumovirus (hmpv), rvfv and lymphocytic choriomeningitis virus (lcmv) in vitro [18, 24, 25, 62, 63] . it was shown that obatoclax inhibited the viral endocytic uptake by targeting the cellular mcl-1 protein [24] . emetine is an antiprotozoal drug. it is administered by intramuscular or deep subcutaneous injection in a dose of 1 mg/kg/day (maximum 60 mg/day) for 10 days [64] . emetine is also used to induce vomiting. in addition, it possesses antiviral effects against zikv, ebov, rabv, cytomegalovirus (cmv), hcov-oc43, hsv-2, ev1, hmpv, rvfv, fluav, hiv-1 and sars-cov-2 [17, 18, 36, [65] [66] [67] [68] [69] (https://doi.org/10.1101/2020.03.25.008482). emetine was proposed to inhibit viral polymerases, though it could have some other targets [70] . homoharringtonine is an anticancer drug that is indicated for the treatment of chronic myeloid leukemia (2 mg/m 2 iv daily × 7). it also possesses antiviral activities against hepatitis b virus (hbv), mers-cov, hsv-1, ev1, vzv and sars-cov-2 in vitro [18, 36, [71] [72] [73] [74] . homoharringtonine binds to the 80s ribosome and inhibits viral protein synthesis by interfering with the chain elongation [72] . salinomycin is an orally bioavailable antibiotic that is used against gram-positive bacteria in animal husbandry (0.2 mg/kg body weight (bw) po). it also inhibits fluav, respiratory syncytial virus (rsv) and cmv infections [75, 76] . salinomycin was proposed to disrupt the endosomal acidification and to block entry of the viruses into cells [77, 78] . our results have uncovered several existing bsaas that could be repositioned to sars-cov-2 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-2 in vero-e6 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 [18, 25, 79, 80] (https://doi.org/10.1101/2020.04.14.039925). 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 16.48 (i.e., 16.48% 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 >10 as true (significant) synergy. thus, the amodiaquine and nelfinavir combination could result in better efficacy and decreased toxicity for the treatment of sars-cov-2 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-2 options on the freely accessible website (https://sars-coronavirus-2.info). the website is updated regularly and incorporates novel anti-sars-cov-2 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-19 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-2 pandemic, as well as future viral outbreaks. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/12/6/642/s1: table s1 : compounds, their suppliers, catalogue numbers and aucs. table s2 . results of neutralization and elisa assays. figure s1 . propagation of hcov-19/norway/trondheim-e9/2020 in cell cultures. figure s2 . effects of temperature and uv radiation on the infectivity of the hcov-19/norway/trondheim-e9/2020 strain. figure s3 . the effects of the serum from patients recovered from the sars-cov-2 infection on the viability of vero-e6 cells infected with 7 sars-cov-2 strains. figure s4 . the effects of nicotine on the viability and morphology of mock-and sars-cov-2-infected vero-e6 cells. figure s5 : the comparison of anti-sars-cov-2 activities of amodiaquine and its analogs. figure s6 . transcriptomic analysis of mock-and sars-cov-2-infected vero-e6 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 mobtt39 (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. 2012, 1, e46 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 bx795 blocks herpes simplex virus type 1 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-1 entry inhibitors targeting gp41 pocket approved antiviral drugs over the past 50 years the convalescent sera option for containing covid-19 effectiveness of convalescent plasma therapy in severe covid-19 patients 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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-19 using autodock the anti-hiv drug nelfinavir mesylate (viracept) is a potent inhibitor of cell fusion caused by the sarscov-2 spike (s) glycoprotein warranting further evaluation as an antiviral against covid-19 infections nelfinavir inhibits maturation and export of herpes simplex virus 1 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 2 replication and infectivity a multicenter phase i/ii study of obatoclax mesylate administered as a 3-or 24-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 33669) 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-1 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 2 function identification of antiviral drug candidates against sars-cov-2 from fda-approved drugs jnj872 inhibits influenza a virus replication without altering cellular antiviral responses in vitro testing of combined hydroxychloroquine and azithromycin on sars-cov-2 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-19 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-2.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-284322-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: 2010-07-27 journal: bmc microbiol doi: 10.1186/1471-2180-10-201 sha: doc_id: 284322 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 39 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 2 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) [1] . 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 [2, 3] . 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 [1] . 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 [4] . 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 [5] [6] [7] . pospischil and wood [7] 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 1978 by pensaert and debouck [8] , 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 50-100 nuclei or more. typical features of syncytial cells were growth, fusion and detachment from cell layers after they had reached a certain size [9] . biomolecular studies revealed major genomic differences between cell culture-adapted (ca)-pedv and wild type virus [10, 11] . cell culture model of co-infection with ca-pedv and chlamydia has been established recently [12] 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 1b) . syncytia were categorized into small (2-15 nuclei), medium (16-30 nuclei) and large (more than 30 nuclei). in single infection experiments, syncytia at 24 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 1) . 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 39 h post infection ( figure figure 1 morphology of chlamydia pecorum mono-and coinfection with pedv. a) vero cells were infected with chlamydia pecorum 1 moi for 39 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 50 μm 2 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 = 0.0044. 1c &2a). in general, chlamydial inclusions were smaller and had more variable forms in chlamydia pecorum than in chlamydia abortus single infections. infectivity was almost 100% 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 1-10 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 2b ). in contrast, dual infections with ca-pedv and chlamydia pecorum resulted in the exclusive production of aberrant inclusions containing between 2-50 abs. chlamydial inclusions in viral syncytia grew even larger than in non-viral infected vero cells. overall, no normal chlamydial inclusions were observed (figure 1a &1b) . 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 2 and all inclusions were assembled into groups covering 50 μm 2 and multiples of this area. the average frequency of chlamydia pecorum inclusions between 100 μm 2 and 400 μm 2 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 = 0.0132. 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 1d ) as analyzed by t-test (p = 0.0044). 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 1a &1b ). 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 0-200 μm 2 was significantly (p = 0.0132) reduced under virus infection but the amount of medium sized and big inclusions 300 -700 μm 2 was increased ( figure 2c ). 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 2b ). 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 [2, 13, 14] . thus, chlamydial ultrastructure in single and co-infected cells was compared by transmission electron microscopy (tem). at 24 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 50 to 130 nm (data not shown). at 39 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 3a and 3b) . 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 2 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 3c ), whereas dual infections with ca-pedv and chlamydia pecorum resulted in the exclusive production of aberrant inclusions consisting of 2-50 abs (figure 3d ). neither chlamydial inclusions nor ca-pedv virions were visible in mock-infected cells. previous studies have demonstrated that chlamydial persistent forms are non-infectious [2] . 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 40 h of re-infection in three independent experiments for chlamydia abortus (figure 4a ) and for chlamydia pecorum (figure 4b ). 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 = 0.0145) (figure 4b ). in chlamydia abortus reinfection analysis, several ebs could still be observed in spite of the co-infection with ca-pedv (figure 4a 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 1) . 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 [12] 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. (2006) [15] . in that study, it was shown that chlamydia trachomatis enters a viable but non-cultivable, persistent state with herpes simplex virus type 2 (hsv-2) 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 [16] . in the study by deka et al. (2006) [15] , hela monolayers were first infected with chlamydia trachomatis and 24 h later with hsv-2. in our study, the optimal experimental protocol for co-infection procedure was developed, based on our own earlier study [12] , 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 [12] , 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 14-15 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 (1992) [17] suggested that strain 1710s 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) [18] . 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 cal10 in l cells showing aberrant chlamydial stages were published by matsumoto and manire [13] . 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., 2004) [1] 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 1710s is an original swine isolate whereas chlamydia abortus strain s26/3 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 [19] ; thus, this cytokine group well known to be capable of inducing in vitro persistence in chlamydia pneumoniae [1] , 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 pd5, pk 15, and hrt18 cell lines [9] . 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 [15] has been recently been shown not to be mediated by any known persistence inducer or anti-chlamydial pathway recently [20, 21] . instead, it was hypothesized by the authors that hsv-2 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-2. finally, it was concluded that the interaction of hsv glycoprotein d with the host cell surface is crucial to trigger chlamydial persistence [22] . 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-2 and chlamydia trachomatis occurs in vivo; thus, the in vitro model described by deka et al. (2006) [15] 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 [23] 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. [23] 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, 25 mm hepes, without l-glutamine (gibco, invitrogen, carlsbad, ca) and supplemented with 10% fetal calf serum (fcs) (bioconcept, allschwil, switzerland), 4 mm glutamax-i (200 mm, gibco) and 0.2 mg/ml gentamycin (50 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 24 h incubation period after the infection with ca-pedv, respectively. incubation medium was prepared as gm without gentamycin, freshly supplemented with 1 μg/ml cycloheximide (sigma, buchs sg, switzerland), and used after an infection for estimation of the chlamydial titer (ifu determination). vero 76 cells (african green monkey kidney cells, crl 1587 american type culture collection) were seeded on round plastic coverslips (13 mm diameter, bibby sterilin, stone, uk) and cultured in gm without gentamycin at 37°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 s26/3 (ovine abortion strain, kindly donated by dr. g.e. jones, moredun research institute, edinburgh, gb) and chlamydia pecorum 1710s (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 1:2 in sucrose-phosphate-glutamate (spg) medium and stored at -80°c. yolk sac-derived chlamydiae were then propagated in hep-2 cell (atcc ccl-23) monolayers and elementary bodies (ebs) were harvested and purified by disruption of hep-2 cell monolayers with a cell scraper, sonication and centrifugation over a renografin density gradient as described elsewhere [24] . eb suspensions were stored in sucrosephosphate-glutamic acid buffer at -80°c, after which viable titers were established using standard methods. moi of 1 was used for chlamydial monoinfection and mixed infection, respectively. ca-pedv strain cv777 (kindly provided by prof. dr. m. ackermann, institute of virology, university of zurich) was propagated as previously described [9] . the virus (10 5,5 tcid 50 /ml) was used undiluted (1 ml 10 5,5 tcid 50 /ml). vero cells, an african green monkey kidney cell line (atcc crl 1587), were used for all infection experiments. they were propagated in gm without gentamycin at 37°c in an atmosphere of 5% co 2 . 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 1 for chlamydia and an infective dose of 1 × 10 5,5 tcid 50 /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 1. all coverslips were centrifuged at 1000 × g for 1 h at 25°c. timepoint 0 (t 0 ) was defined after centrifugation and supernatant was replaced subsequently by incubation medium. infected monolayers were then incubated for 14 h at 37°c (t 0 -t 14 ). all cell layers for dual infections or ca-pedv monoinfection were exposed to a ca-pedv suspension (1 × 10 5,5 tcid 50 ), the samples were centrifuged again for 1000 × g for 1 h at 25°c and incubated for 24 h at 37°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 (-20°c) for 10 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 204, kindly provided by prof. dr. m. ackermann, institute of virology, university of zurich), diluted 1:4 in pbs supplemented with bsa, and an alexa fluor 594-conjugated secondary antibody (goat anti-mouse, 1:500, 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 488-conjugated secondary antibody (goat anti-mouse, 1:500, molecular probes). dna was labeled with 1 μg/ml 4', 6-diamidin-2'-phenylindoldihydrochlorid (dapi, molecular probes). all staining procedures were conducted at room temperature. antibody incubations were carried out for 1 h (primary antibodies) or 45 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 2.5% glutaraldehyde (electron microscopy sciences, ft. washington, usa) for 1-2 h, and processed by routine methods for embedding in epoxy resin (fluka). appropriate areas for ultrastructural investigation were selected using semithin sections (1 μm) stained with toluidine blue (fluka, buchs sg, switzerland). ultrathin sections (80 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 cm10 electron microscope. at 39 h after chlamydial infection, monolayers were scraped into 1 ml of cold infection medium, pelleted and resuspended in 1 ml of fresh medium. infected host cells were lysed by sonication and centrifuged (500 g for 5 min) to remove pellet cell debris. supernatants were centrifuged once (4,000 g for 60 min). final eb pellets were resuspended in 200 μl of spg and used to infect vero cells plated on glass coverslips in duplicate in dilution series. all coverslips were centrifuged at 1000 × g for 1 h at 25°c. after centrifugation, the vero cells were refed with medium containing 1 μg/ml cycloheximide and subsequently incubated for 40 h at 37°c. fixation and staining of chlamydia, ca-pedv and dna was performed as described above. the number of inclusions in 20 random microscopic fields per sample was determined using a leica fluorescence microscope at a magnification of 200 ×. 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 10 6 cells as described by deka et al., 2006 [15] . 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-2 cells chlamydia trachomatis enters a viable but non-cultivable (persistent) state within herpes simplex virus type 2 (hsv-2) 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 3-mediated antiviral response in a cell line deficient for ifn production an early event in the herpes simplex virus type-2 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-2 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 1 institute of veterinary pathology, vetsuisse faculty, university of zurich, zurich, switzerland. 2 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-333208-tibtngy8 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: 2016-04-26 journal: plos one doi: 10.1371/journal.pone.0154366 sha: doc_id: 333208 cord_uid: tibtngy8 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 ba71v 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 ifitm1, 2 and 3 reduced virus infectivity in vero cells, with ifitm2 and ifitm3 having an impact on viral entry/uncoating. the role of ifitm2 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 ifitm2 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 [1] . this group of proteins is present across a wide range of species: from amphibians, fish and birds to mammals. ifitms in humans were identified 26 years ago as interferon-stimulated genes upon induction of type-i and type-ii ifn [2, 3] . human ifitm1, ifitm2 and ifitm3 are expressed in almost every cellular type, whereas ifitm5 is expressed primarily in osteoblasts, as it is required for bone mineralization [4] . ifitms are found mainly distributed at the plasma membrane and/or at endosomal membranes. the ifitm1, 2, 3 and 5 genes are clustered on chromosome 11, and they encode for relatively small proteins (about 130 amino acids) with both extra-cytoplasmic termini separated by two transmembrane domains (tm1 and tm2) and a cytoplasmic loop (cil) [3] [5] . tm1 and the cil are well conserved between the ifitm proteins and a large group of members of the cd225 protein family. ifitm 1, 2, and 3 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) [6] , severe acute respiratory syndrome coronavirus (sars cov) and hepatitis c virus (hcv) [7] . 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) [4] . little is known about the ifitm-mediated antiviral activity against dna viruses. only ifitm1 has been recently described to inhibit rana grylio virus (rgv), a frog/fish iridovirus, at the entry stage [8] . on the other hand, ifitm1, 2 and 3 have been reported not to affect the replication of other dna viruses, such as human papillomavirus (hpv), human cytomegalovirus (hcmv) and adenovirus 5 (ad5) [9] . 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 [4] . 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 [10] . asfv infection is strongly dependent on the endocytic pathway [11, 12] , 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 [13] . an epidemic outbreak is currently affecting east europe and is slowly spreading between neighboring countries [14] [15] [16] . we previously reported that asfv enters into the host cell by dynamin-dependent and clathrin-mediated endocytosis [12, 17] . 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 ba71v isolate. vero cells were obtained from the american type culture collection (atcc, richmond, va, usa) and maintained in dulbecco modified eagle medium supplemented with 5% fetal bovine serum (fbs), 100 lu/ml penicillin, 100ug/ml streptomycin and 2mm l-glutamine at 37°c at 5% co 2 . cells were pretreated with 1,000 or 10,000 u/ml of universal type-i ifn (pbl assay science) for 24 h, as indicated. the tissue culture-adapted asfv isolate ba71v was used in most experiments [18] . for flow cytometry analyses, a recombinant virus expressing the viral protein p54 fused to the green fluorescent protein (gfp) was used (b54gfp) [19] . preparation of viral stocks, titrations and infection procedures were carried out in vero cells as previously described [18] . when synchronization of viral infection was required, the adsorption phase took place at 4°c to allow viral attachment to the cell surface but impeding its internalization. when indicated, asfv was semi-purified by sucrose cushion (40%) in pbs at 40,000xg for 50 min at 4°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. 293t cells were transfected at 100% confluency using lipofectamine 2000 (life technologies) with opti-mem (life technologies) in 10-cm 2 plates. plates were previously pretreated with poly-l-lysine (sigma-aldrich) at a final concentration of 0.1 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 48 h and 72 h postransfection. cell debris was removed by brief centrifugation at 1,000 rpm for 5 min and cleared supernatants were 0.22 μm-filtered and stored at -80°c until use. sub-confluent vero cells were transduced with the pseudotyped lentiviruses expressing the gene of interest and supplemented with 1 μg/ml of polybrene (emd millipore). 24 h later, transduced cells were selected by adding 8 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 4% paraformaldehyde in pbs for 15 min at room temperature (rt). following cell fixation, aldehyde fluorescence was quenched by incubation of cells with 50 mm nh 4 cl in pbs for 10 min. then, cells were permeabilized with pbs-0.1% triton x-100 or saponin (sigma) for 10 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-3 (molecular probes) in pbs at a 1:1,000 ratio for dna staining. after washing, coverslips were finally mounted on glass plates and cells were observed under a leica tcs sp2-aobs confocal microscope (leica-microsystems, wetzlar, germany) using a 63x immersion oil objective. to detect cholesterol distribution we used filipin staining (sigma), as previously described [20] . cholesterol was visualized in a conventional leica dm rb microscope by combining a 63x 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 8.0. the primary antibodies used for immunofluorescence assays included the following: rabbit polyclonal antibodies to ifitm1, ifitm2 and ifitm3 (proteintech), 1:200; anti-asfv p30 mouse monoclonal antibody, 1:100 (kindly given by dr. j.m. escribano, inia); asfv mouse monoclonal antibodies anti-p72 (clone 1bc11 for immunofluorescence 1:1,000 or clone 18bg3 for wb 1:2,000) and anti-p150 (clone 17ah2, ingenasa), 1:1,000; mouse monoclonal to cd63 (developmental studies hybridoma bank, university of iowa, clone h5c6), 1:200; rabbit polyclonal to lamp1 (abcam), 1:50; rabbit polyclonal to eea1 and rab7 (cell signalling), 1:50. secondary antibodies were purchased from molecular probes and diluted 1:200. specificity of labeling and absence of signal crossover were determined by examination of single labeled control samples. cells were harvested in sample buffer laemmli 2x concentrate (sigma). then, the samples were incubated for 5 min at 95°c and resolved by sds-page in 12% or 7% 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 ifitm1, ifitm2 and ifitm3 (proteintech), 1:2,000; anti-asfv p30 mouse monoclonal antibody, 1:500; anti-asfv p72 mouse monoclonal antibody (clone 1bc11, ingenasa), 1:1,000, and mouse monoclonal to tubulin (sigma-aldrich), 1:2,000. as secondary antibody, anti-mouse igg (ge healthcare) or anti-rabbit igg (bio-rad) conjugated to horseradish peroxidase was used at a 1:5,000 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, 1:2,000. 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) (2x; takara) probe. fluorescent hybridization probes were used to amplify a region of the p72 viral gene, as described previously [21] . dna from cells mock-infected or infected with asfv ba71v at moi of 1 pfu/cell was extracted at 16 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: 250 ng template dna diluted in mq h 2 o to a total volume of 7μl, 1 μl oligonucleotide oe3f (50 pmol), 1 μl oligonucleotide oe4r (50 pmol), 10 μl pcr premix ex taq(tm) (2x; takara), 1 μl taqman probe se2 (5 pmol) [21] . 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 7500 fast real-time pcr system (applied biosystems) with the following parameters: 1 cycle at 94°c for 10 min, 45 cycles at 94°c for 15 s, and 45 cycles at 58°c for 1 min. to study virion decapsidation in asfv, a protocol was adapted from a previous publication [22] . briefly, stable cell lines vero-ifitm1, ifitm2, ifitm3 or control cells containing the empty vector were infected at moi of 10 pfu/cell after viral synchronization at 4°c for 90 minutes to enable virus attachment to the cell but restricting viral entry. infection was allowed to proceed for 75 minutes at 37°c, 5% co 2 . cells were then washed with cold pbs 1x and treated with 0.05% trypsin-edta (gibco) for 10 minutes at 37°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 p72 and the viral core protein p150, and staining was analyzed by confocal microscopy. decapsidated virions were single labeled for p150 and were counted for each condition and normalized to the total number of fully encapsidated virions which were double labeled for p72 and p150. stable cell lines vero-ifitm1, ifitm2, ifitm3 or control cells containing the empty vector were infected with ba71v or b54gfp at the indicated moi. recombinant b54gfp is a recombinant asfv expressing green fluorescent protein as a fusion protein of viral p54 [19] . samples infected with b54gfp at 16 hpi were just fixed and washed with facs buffer three times before analysis. vero cells infected with ba71v at 6 hpi or 16 hpi (early or late postinfection times respectively), were harvested by trypsinization, washed with facs buffer (containing pbs, 0,01% sodium azide, and 0,1% bovine serum albumin), fixed and permeabilized with perm2 (bd science) for 10 min at rt and finally incubated with specific primary antibodies against p30 and p72 for 30 min at 4°c. the secondary antibody used was phycoerythrin (pe) conjugated (dako) 1:50 diluted in facs buffer for 30 min at 4°c. after repeated washes, 10,000 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 instat3 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 ( ããã p<0.001, ãã p<0.01 and ã p<0.05). to determine the effect of interferon on asfv infection, vero cells were pretreated with 1,000 or 10,000 u/ml of universal type-i ifn (pbl assay science) and 24 h later, they were infected with recombinant asfv b54gfp at a moi of 5 pfu/cell (fig 1) . viral infection was quantified by analyzing the number of gfp-positive cells by flow cytometry at 16 hpi (fig 1a) . 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 1a) . 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 [23, 24] . then, we analyzed ifitms expression and distribution in vero cells upon ifn treatment. to this end, cells were incubated with either 1,000 or 10,000 u/ml of universal type-i ifn for 24 h and ifitms 1, 2 and 3 distribution was analyzed by confocal microscopy ( fig 1b) . although basal levels of ifitm2 and 3 were detected prior to treatment with to further analyze the expression of ifitm1, 2 and 3 upon ifn treatment, vero and 293t cells were incubated with universal type-i ifn for 24 h and analyzed by wb (fig 2) . while ifitm1 and ifitm2 were induced by ifn in both cell lines (fig 2a and 2b) , ifitm3 showed the highest induction (fig 2c) . in order to analyze the possible impact of ifitms in asfv infection, we generated vero cells stably expressing the human ifitm1, 2, 3 (hereinafter referred to as vero-ifitm1, 2 or 3 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 8 μg/ml of puromycin. once these cells were established, the expression of different ifitm proteins was confirmed by wb analysis ( fig 3a) . as shown in the corresponding wb densitometry (fig 3b) , the highest expression levels within the ifitm family members corresponded to ifitm3, followed by ifitm2 and ifitm1. after assessing the expression levels of the vero-ifitm cells, we next wanted to ascertain the subcellular distribution of each ifitm. expression of ifitm1, 2 and 3 in vero-ifitm cells was compared with the distribution of ifitms in vero cells with the empty vector. confocal microscopy experiments revealed that, ifitm1 was mainly distributed at the plasma membrane and to a lesser extent in perinuclear compartments, resembling endosomal structures (fig 3c, lower left panel) , while endogenous ifitm1 was barely detected in vero cells containing the empty vector (fig 3c, upper left panel) . in vero-ifitm2 cells, overexpression led to a high accumulation of the protein in the perinuclear region, colocalizing with vesicular structures that resembled endosomes (fig 3c, medium lower panel). consistent with fig 1b, there was a significant expression of endogenous ifitm2 in control vero cells containing the empty vector, which displayed a mitochondrialike pattern together with vesicular-like structures. finally, overexpressed ifitm3 was found predominantly accumulated in the perinuclear region, showing a pattern consistent with localization at clustered endosomal structures ( fig 3c, lower right panel) . endogenous ifitm3 was barely detected in vero cells containing the empty vector (fig 3c, upper right panel) . analysis of endogenous ifitm2 expression in control vero cells suggested a mixed mitochondrial and vesicular pattern. to confirm this observation, we analyzed subcellular localization of ifitm2 using mitotracker red as a specific marker for mitochondria. confocal images revealed a distribution of ifitm2 to mitochondrial structures in cells containing the empty vector (fig 4) . however, in vero-ifitm2 cells, ifitm2 labelling localized to endosomal-like structures and we found few areas of colocalization between ifitm2 and mitochondria (fig 1b) . 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<0.001). an example of a typical facs profile is shown. (b). confocal fluorescence images of ifitm1, 2 and 3 subcellular distribution in untreated vero cells or upon increasing universal ifn concentrations (1,000 or 10,000u/ml) for 24 h. bar = 10μm. (fig 5a) . 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-ifitm1 cells. however, in vero-ifitm2 and ifitm3 cells dispersed distribution changed and endosomes aggregated around the nucleus (fig 5a) . 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 5b) . a total of 30 cells were analyzed for each condition. we concluded that overexpression of ifitm2 and ifitm3 altered endosome distribution by accumulating these vesicles to the perinuclear region, similar to the pattern previously found after ifn treatment of vero cells (fig 1b) and this redistribution might reflect alterations in endo-lysosomal maturation and function. next, we analyzed the colocalization rate between ifitms and endosomal structures. cd63 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 cd63 by immunofluorescence assay revealed a clear colocalization of ifitms and cd63 in vero-ifitm cells, with 75% colocalization for vero-ifitm2 cells and 40% in vero-ifitm1 and in ifitm3 cells (fig 6a) . then, ifitm2, and to a lesser extent ifitm1 and 3, were located primarily in endosomal compartments as indicated by higher colocalization with endosomal marker cd63 (fig 6c) when compared to the empty vector ( fig 6b) . ifitm proteins could reduce curvature of cell membranes for fusion pore formation [24, 25] 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 5) is a characteristic phenotype of an altered cholesterol endosomal efflux [23, 26] . 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-ifitm2 and ifitm3 cells showed intense intracellular cholesterol accumulation at the perinuclear area ( fig 7a) that was absent in control cells containing the empty vector (fig 7b) . this cholesterol accumulation also colocalized with the ifitm-labeled endosomal vesicles. in contrast, in vero-ifitm1 cells, only discrete colocalization areas between ifitm1 and cholesterol were found at the plasma membrane. collectively, these findings indicate that ifitm2 and ifitm3 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 [11] . previous experiments in the laboratory revealed that the inhibition of acidification impaired viral decapsidation and viral particles were retained in rab7-positive late endosomes, thus blocking viral infection progression [11] . given that ifitm restriction could be mediated at the endocytic pathway [1, 4] , 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 [11] . the asf virion is composed of several concentric domains. the viral capsid is formed by major capsid protein p72 organized in capsomers. hence, after decapsidation, p72 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 p150, a core shell protein derived from processed core shell polypeptides [27] . . (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-ifitm2 and 3 cells. graphics depict mean±sd of n = 30 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<0.05; **p<0.01). doi:10.1371/journal.pone.0154366.g005 we used antibodies against the major viral capsid protein p72 to detect viral capsids and against viral core protein p150 to detect viral cores (fig 8) . we analyzed the number of encapsidated viral particles, double labeled for p72 and p150, and compared with the number of successfully decapsidated viral cores positive for p150 at 75 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-ifitm2 cells when compared to control cells containing the empty vector (fig 8b) . 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 8a) . the percentage of decapsidated virus severely decreased under ifitm2 expression. however, ifitm3 expression produced an accumulation of virions leading to higher numbers of total virions (fig 8c) . 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-ifitm2 and ifitm3 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 16 hpi (fig 9a) . ifitm overexpression induced a consistent reduction in infectivity as measured by early protein p30 expression by flow cytometry (fig 9b) . however, these reductions were even more marked when analyzing infected cell percentages by late p72 protein expression (fig 9c) . finally, we also analyzed viral protein expression by wb (fig 9d-9f) . the expression of protein p30 at 6 hpi ( fig 9d) and p72 at 16 hpi (fig 9e) 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 [6] , 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 [8] . asfv ba71v infection in vero cells is significantly affected upon ifn treatment [28, 29] . 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 a276r, which negatively regulates the induction of ifn through targeting irf3 in a nfκb-independent manner [30] . another example is the asfv gene i329l, which codifies for a viral tlr3 homolog with inhibitory activity against ifn [31] . 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 [32] . 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 ifitm2 expression in the absence of ifn induction showed colocalization with mitochondrial structures. interestingly, ifitm2 underwent vesicular pattern redistribution around the cell nucleus when overexpressed and upon ifn treatment as well. our characterization of the cellular distribution of ifitm1, 2 and 3 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 ifitm1 at the plasma membrane and early endosomes, and of ifitm2 and ifitm3 in late endosomes and lysosomes [33, 34] . interestingly, overexpression of ifitm1 has been recently described to delay the proteolytic degradation of human papilloma virus (hpv) capsids in keratinocytes [9] . this, however, did not affect the replication of the virus. our analysis of asfv ba71v uncoating correlated the expression of ifitm2 with a decrease in the numbers of decapsidated asf virions in vero cells. this suggests a possible role for ifitm2 in inhibiting asfv exit from late endosomes. in contrast, ifitm3 did not modify the ratio between encapsidated and decapsidated virions. instead, ifitm3 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 ifitm3 affects the release of the virions from the late endosomal compartments. asfv enters into the host cell by dynamin-dependent and clathrin-mediated endocytosis [12, 17] and macropinocytosis [35] . in fact, endosomal pathway integrity is known to be important for asfv infection, both for culture-adapted isolates in cell lines [11] or for virulent and attenuated isolates in primary macrophages [12] . hence, it is not surprising that infection could be impaired by these restriction factors acting at the endosomal membrane. the aforementioned ifitm2-and ifitm3-mediated inhibition of asfv entry has been previously reported in other viruses, including iav, flaviviruses (denv and wnv) [6] , filoviruses (marv and ebov) and coronaviruses (such as sars) [34] . in contrast, infection with alphaviruses, arenaviruses and mlv (a retrovirus) seems to be unaffected by ifitm protein expression [4] . in general, ifitm-mediated viral inhibition has been related to impaired viralhost membrane fusion subsequent to viral binding and endocytosis [33, 34] . ifitm3 has also been reported to modulate the fluidity and the bending modulus of the cell membrane, thus making it resistant to viral fusion machinery [36] . 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 ifitm2 and ifitm3 altered the normal distribution of early and late endosomes and lysosomes. however, this alteration was not found in the presence of ifitm1. a collapse of endosomes to the perinuclear area is also a characteristic phenotype of an alteration of endosomal cholesterol efflux [26] . 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 [37] . 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 [36] . the second model suggests that ifitms can induce the accumulation of cholesterol in the endosomal membrane and membrane fusion defects [38] disturbing intracellular cholesterol homeostasis that finally blocks the viral release into cytosol [23] . ifitm2 and ifitm3 have been previously reported to alter the cholesterol homeostasis at the late endosome, leading to cholesterol accumulation and blocking the viral release [23] . in the present study, we have described accumulation of cholesterol upon overexpression of ifitm2 and ifitm3. 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 [37] . 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-1 virions [39] . we have performed this study in a cellular system using the cell-adapted ba71v 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 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cord-351525-306syrrn 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 hku2-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: 2019-11-26 journal: j virol doi: 10.1128/jvi.01448-19 sha: doc_id: 351525 cord_uid: 306syrrn outbreaks of severe diarrhea in neonatal piglets in guangdong, china, in 2017 resulted in the isolation and discovery of a novel swine enteric alphacoronavirus (seacov) derived from the species rhinolophus bat coronavirus hku2 (y. pan, x. tian, p. qin, b. wang, et al., vet microbiol 211:15–21, 2017). 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 556:255–258, 2018). 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, c57bl/6j 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-hku2-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-hku2-origin cov. different animal species were tested for susceptibility to sads-cov treated with or without trypsin ( table 1) . as a brief summary of the results, 21 of the 24 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 264.7. first, cpe was examined by inverted light microscopy at 48 hours postinfection (hpi), and scores are shown in table 1 . as the 293t, nih/3t3, cho, brl-3a, and nrk-52e 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-3a cell lines without trypsin, and prominent cpe appeared in or was enhanced with trypsin in marc-145, cos-7, bsc-40, vero, st, pk15, llc-pk1, and bhk-21 cell lines ( table 1) . 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. 1) , revealing the same range as seen by cpe in the different cell lines (data not shown). syncytium formation was prominent in huh-7, vero, and bhk-21 cells, whereas in mdck, bfk and raw 264.7 cells, the antigen expression was much less prominent than in the other cell lines (fig. 1a , 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-145, llc-pk1, and ipec-j2 cells suggested only a limited infection. next, viral load in the culture supernatants was detected over 5 days postinfection (dpi) by reverse transcriptase quantitative pcr (qrt-pcr) ( fig. 2a to c). a higher mean viral load was detected by qrt-pcr after trypsin treatment in hepg2, hela, marc-145, cos-7, bsc-40, vero, llc-pk1, ipec-j2, bhk-21, and df-1 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-7 and tb-1 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 superto 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 2 h and subsequently replaced with mm (a); "pretrypsin" treatment, inoculated with sads-cov diluted in mm containing 5 g/ml trypsin (mmt) for 2 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 12, 24, 36, 48, 72, and 120 hpi for viral load detection by a qrt-pcr assay targeting the viral n gene. data are expressed as the mean viral load (log 10 copies/l) ϯ standard deviation (sd), and all experiments were performed in triplicate. the 293t, cho, brl-3a, and nrk-52e cell lines did not survive in the presence of trypsin. (d) infectious titers (tcid 50 /ml) of sads-cov secreted from hela, vero, tb-1, bhk-21 pk-15, and mdck cells were determined on vero cells. natants in vero cells (fig. 2d ). unlike in mdck cells, sads-cov infection of hela, vero, tb-1, bhk-21, and pk-15 cells was productive, with hela cells showing the greatest susceptibility (fig. 2d) . wild-type c57bl/6j 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 6-to 8-week-old wild-type b6 mice with 5 ϫ 10 5 50% tissue culture infective dose (tcid 50 ) of sads-cov by the per oral (p.o.) or intraperitoneal (i.p.) route and monitored them for 28 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 1, 3, 5, 7, 14, 21, and 28 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 7 or 14 dpi and thereafter (fig. 3a) . 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. 3b ). 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 1 to 3 dpi, and much lower viral rna at 21 to 28 dpi in the colon than that of the p.o. route (fig. 3c) . however, this replication in the large intestine did not translate into higher shedding, as hardly any viral genomes were detected, even at 1 dpi in the fecal samples collected from i.p.-infected mice (fig. 3f ). on the contrary, significantly more virus was detected in the feces of p.o.-infected mice at 1 and 3 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 21 dpi (fig. 3d ). more importantly, the virus was not cleared from this tissue by 28 dpi in the i.p.-infected group and by 14 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 1 to 3 dpi, and no virus was detectable at later time points (fig. 3d ). 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. 3e ). igg antibody levels after 7 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. 3g) . 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 5 ϫ 10 5 tcid 50 of sads-cov at 3 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. 4a ). staining of tissue sections from mock-infected mice were used as a control (fig. 4b ). in addition to dsrna, we also used rabbit polyclonal antibodies (pabs) to detect the expression of the viral structural protein (m) or nonstructural protein (nsp3-ac). at 3 dpi, anti-m or anti-ac staining was observed in the white pulp around the lymphatic nodules (fig. 4c) , similar to the localization of dsrna staining (fig. 4a ). 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. 3) . next, sads-cov infection was quantified in the spleen using flow cytometry. we inoculated b6 wild-type mice with 5 ϫ 10 5 tcid 50 of virus either i.p. or p.o. and extracted the bulk immune cells from the spleen of infected animals at 3 dpi. the flow cytometry method was first validated in vero cells infected with sads-cov at a multiplicity of infection (moi) of 0.01, followed by staining with a pab against the n or ac protein at 24 hpi (fig. 4d ). as the anti-ac pab exhibited optimal intracellular staining for viral signals (fig. 4d) , it was used to determine the percentage of infected splenocytes. there were approximately 1.5-and 2.5-fold increases of total splenocytes positive for virus replication after p.o. and i.p. inoculation, respectively (fig. 4e , left; fig. 4f ), with a significant increase in the total number of ac-positive splenocytes in i.p.-infected mice compared with that of p.o. (fig. 4e , right). these data are consistent with the significantly lower viral loads in the spleen at 1 and 3 dpi in p.o.-inoculated mice (fig. 3d ), 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 100-mm dishes, and infected with 1 ϫ 10 5 tcid 50 of sads-cov. we observed clusters of infected cells that appeared to have been engulfed by phagocytes (fig. 4g , middle), and the structural n protein was shown in the cytoplasm of infected cells by confocal microscopy (fig. 4g , middle and right). the percentage of infected cells was quantified by flow cytometry using anti-ac pab, revealing a nearly 2-fold increase in the splenocytes positive for viral signals (fig. 4h) , 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 1 ϫ 10 5 tcid 50 of sads-cov, and culture supernatants were harvested at 0, 12, 24, 48, and 72 hpi. active viral replication was confirmed, with a 1.5-log time-dependent increase in genomic rna equivalents, plateauing from 24 to 72 hpi (fig. 4i ). these data suggest that although only ϳ2% 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 3 dpi, and the extracted cells were costained with antibodies against sads-cov-ac and each of four cell surface markers (anti-cd19 for b cells, anti-cd4 for t cells, anti-cd11/c ϩ for dcs, and anti-f4/80 ϩ for macrophages) using flow cytometry (fig. 5a ). the percentage of infected cd11/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-cd11/c ϩ in splenic sections. as expected, dsrna staining overlapped with the cd11/c surface marker on the edges of lymphatic follicles (fig. 5b) , whereas no viral signals were seen in the mock-infected control (fig. 5c ). similar patterns of costaining were detected by m and ac antibodies (fig. 5d ). to gain insight into the relative quantity of dcs compared with other undefined target cells, cells positive for dsrna and cd11/c were counted in 10 to 15 different microscope fields of spleens from 3 infected mice (fig. 5e) , showing that 61.76% of sads-covinfected cells were dcs (fig. 5f ). 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. 1 and 2) , were chosen as a potential candidate. there are four known types of functional cov protein receptors, including angiotensin converting enzyme 2 (ace2) for sars-cov (18), dipeptidyl peptidase 4 (dpp4) for mers-cov (19) , aminopeptidase n (apn) for tgev (20) and pdcov (21, 22) , and mouse carcinoembryonic antigen-related cell adhesion molecule 1a (mceacam1a) for mouse hepatitis virus (mhv) (23) . to test whether one of these molecules serves as the sads-cov receptor, we attempted to inoculate nonsusceptible mdck cells overexpressing porcine apn, human dpp4, mouse ceacam1a, or human ace2 with sads-cov, but none of them allowed infection, as staining with anti-sads-cov-n pab was negative (fig. 6a) . meanwhile, the expression of each receptor in mdck cells was confirmed by ifa (fig. 6a) and western blot analysis (fig. 6b) 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. 6c) . next, we demonstrated that mdck cells can confer sads-cov replication competency by transfection of a sads-cov/seacov infectious cdna clone established recently (24) , as simultaneous expression of nsp3-ac and n proteins were clearly detected by ifa (fig. 6d) . 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. 6e) , 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. (14) ; however, the hela cell line itself was most susceptible to sads-cov infection in the present study (fig. 2d) . in order to assess the potential species barriers of sads-cov infection, a cell line susceptibility study was first conducted using 24 different cell lines. as sads-cov probably originated from a bat sadsr-cov (14) derived from hku2-cov identified in rhinolophus sinicus (chinese horseshoe bats) (12), we commenced testing viral susceptibility in two available bat cell lines, namely, bfk from myotis daubentonii (25) and tb-1 from tadarida brasiliensis. although bfk cells did not support sads-cov replication, it replicated efficiently in tb-1 cells (fig. 1a and fig. 2) , 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-21, which is not susceptible to other known human covs, such as sars-cov and mers-cov (26, 27) , as well as three swine enteric covs, namely, pedv, pdcov, and tgev (21) . 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 b6 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 (11) . also, there was a lack of robust viral replication in the intestines during infection, and no tissue damage was detected throughout the intestines ( fig. 3b 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 28-day period (fig. 3d) . the phenotype was also consistent with the replication kinetics in extracted splenocytes in vitro, in which viral genomic rna peaked and plateaued at 72 hpi ( fig. 4g 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. 3b to e) , suggesting the important role of mucosal immunity for controlling early infection in sads-cov in mice. it should be noted that mice (c57bl/6j 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 (28) . 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. 5) . 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 (29) . in addition, based on the fact that sars-cov spike-pseudotyped hiv-based vectors can efficiently transduce human dcs, kobinger (31) . 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 hku2, including sads-cov, possesses unique s genes closely related to the betacoronavirus (␤-cov), in a manner similar to some globally distributed rodent ␣-covs (11, 32, 33) , implying an unknown evolutionary connection between the bat ␣-cov hku2 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 hku2-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 (34) . 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 (35) . 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 1 and fig. 1) . 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 (36) . 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. 6 ) 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-01/2017 at passage 10 was used in all experiments and cultured in vero cells (24) . the virus was passaged serially using the culture supernatant to infect fresh vero cells at a multiplicity of infection (moi) of 0.1, and viral titers were determined in vero cells by endpoint dilution as the 50% tissue culture infective dose (tcid 50 ). rabbit polyclonal antibodies (pab) against the membrane (m), nucleocapsid (n), and the nonstructural protein 3 (nsp3) acidic domain (ac) of sads-cov were generated in-house and validated in sads-cov-infected vero cells (24) . 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 j2; catalog number j2-1702, 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 1) , including human (huh-7, hepg2/c3a, 293t, a549, and hela), monkey (marc-145, cos-7, bsc-40, and vero), swine (st, pk15, llc-pk1, and ipec-j2 [37] ), bat (bfk [25] and tb-1), canine (mdck), mouse (nih/3t3 and raw 264.7), hamster (bhk-21 and cho), rat (brl-3a and nrk-52e), and chicken (df-1) 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 10% (vol/vol) fetal bovine serum (fbs; biological industries), 100 u/ml penicillin, and 100 u/ml streptomycin under 37°c, 5% co 2 , and water-saturated humidity conditions. to determine viral susceptibility, each cell line was cultured at 70% confluence in 12-well plates with maintenance medium (mm) containing dmem, 0.3% tryptose phosphate broth (tpb), and 1% penicillin-streptomycin or mm with addition of 5 g/ml trypsin (mmt) (catalog number t7186-50tab; 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 0.01 for 2 h. nonattached viruses were removed by washing the cells three times with dmem, and cell monolayers were subsequently incubated in mm or mmt at 37°c for 5 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 12, 24, 36, 48, 72, and 120 hours postinfection (hpi) for one-step quantitative rt-pcr analysis. cell cultures were examined for cytopathic effects (cpes) and immunofluorescence assay at 48 to 72 hpi. ifa for cell line susceptibility. different cells infected with sads-cov in 12-well plates were washed twice with pbs and fixed in 4% paraformaldehyde in pbs and then permeabilized with 0.1% triton x-100 in pbs. cells were then incubated with the rabbit anti-sads-cov-m pab at 1:5,000 dilution for 1 h at 37°c, washed with pbs, and stained with the alexa fluor 488-conjugated goat anti-rabbit secondary antibody (thermo fisher scientific, usa) at 1:1,000 dilution. after incubation for 1 h at 37°c, the cells were washed with pbs, stained with 4=,6-diamidino-2-phenylindole (dapi) at 1:1,000 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-28a vector using two unique restriction sites, namely, ndei and xhoi, and then linearized with xhoi. the n gene was in vitro transcribed using the t7 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 5=-ctaaaactagccccaca ggtc-3= and 5=-tgattgcgagaacgagactg-3= and the probe 6-carboxyfluorescein (fam)-gaaacccaa actgaggtgtagcagg-6-carboxytetramethylrhodamine (tamra). samples with a cycle threshold value of ͻ35 were considered positive based upon validation data using the rna standards. mouse infections, tissue harvest, and viral load determination. wild-type c57bl/6j mice (catalog number 000664; 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, 6-to 8-week-old female and male mice were inoculated with 5 ϫ 10 5 tcid 50 (equal to 6 ϫ 10 8 genome copies) of sads-cov, either per oral (p.o.) infection with 25-l inoculum (2 ϫ 10 7 tcid 50 /ml) or intraperitoneal (i.p.) infection with 200-l inoculum (2.5 ϫ 10 6 tcid 50 / ml). for viral load determination in specific tissues, mice were euthanized at 1, 3, 5, 7, 14, 21, and 28 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 2% fbs) by bead beating using sterile zirconium oxide beads (catalog number zrob20; 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 1:100 dilution were detected in wells coated with purified sads-cov virus particles (6.25 ng/well) as antigen. histopathology, immunohistochemistry, and immunofluorescence assay for murine spleen. mice were infected i.p. with sads-cov, and at 3 dpi, spleens were harvested and fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. tissue sections were then deparaffinized and rehydrated in three changes of xylene, 15 min each, dehydrated in two changes of pure ethanol for 5 min, followed by rehydration in an ethanol gradient of 85% and 75% 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 6.0) and maintained at a subboiling temperature for 8 min, were left at 98°c for 8 min, and then incubated again at subboiling temperature for 7 min. after the sections were allowed to cool to room temperature (rt) and were washed three times with pbs (ph 7.4), endogenous peroxidase was blocked by immersion in 3% hydrogen peroxide at rt for 30 min, and sections were again washed with pbs. tissue sections were blocked in 3% bovine serum albumin (bsa) at rt for 30 min and then incubated with a 1:500 dilution of each primary antibody (anti-dsrna mab, anti-sads-cov-m pab, or anti-sads-cov-ac pab) overnight at 4°c. after slides were washed three times with pbs (ph 7.4), they were stained with appropriate secondary antibodies labeled with horseradish peroxidase at rt for 50 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 5 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 cd11c antibody (wuhan servicebio technology) at a 1:200 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 cd11/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 3 dpi, and spleens were removed and placed in 5 ml complete dmem. after the excised spleen was ground through a 100-m cell strainer using the plunger end of a 5-ml syringe, cells were washed with an excess of dmem and centrifuged at 200 ϫ g for 5 min. after cells were resuspended in 3 ml of red blood cell lysis buffer (solarbio life sciences, beijing, china) and incubated at rt for 10 min, 5 ml of dmem was added, and cells were passed through another strainer to remove clumps. after centrifugation at 200 ϫ g for 5 min, the supernatant was discarded, and cells were resuspended in 10 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 cd16 fc block antibody at 1:500 dilution) and incubated on ice. cells in fc block buffer were added to 96-well plates at 1 ϫ 10 6 cells/well. after a 30-min incubation, cells were centrifuged at 200 ϫ g for 10 min, the supernatant was discarded, and pellets were resuspended in a 100-l cytofix/cytoperm solution (cytofix/cytoperm soln kit; bd biosciences, san jose, ca, usa) to fix cells. after incubation on ice for 20 min protected from light, cells were centrifuged at 800 ϫ g for 5 min at 4°c, supernatant was removed without disturbing cell pellets, and cells were washed twice in 150 l of 1ϫ perm/wash buffer. after the addition of 50-l virus-specific primary antibody (anti-dsrna mab, anti-sads-cov-n pab, or anti-sads-cov-ac pab) diluted in 1ϫ perm/wash buffer with 3% bsa and incubation at 4°c for 30 min, cells were centrifuged at 200 ϫ g for 10 min. cells were washed twice in 150 l of 1ϫ perm/wash buffer followed by staining with appropriate secondary antibodies conjugated to alexa fluor 488 (thermo fisher scientific, usa) at 4°c for 30 min. after pellets were centrifuged at 800 ϫ g for 5 min at 4°c and washed with 1ϫ perm/wash buffer, they were resuspended in 0.2 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 100-or 35-mm dishes, and infected with sads-cov at an moi of 0.1. at 48 hpi, splenocytes were harvested and placed in a 15-ml tube, centrifuged at 200 ϫ g for 10 min at 4°c, and analyzed by flow cytometry as described above. infected mouse splenic cells in 35-mm dishes were detected by immunofluorescence assay with anti-sads-cov-n antibodies, and infection supernatants were collected at 0, 12, 24, 36, 48, and 72 hpi for one-step quantitative rt-pcr analysis. facs analysis of splenocytes with cell marker staining. mice infected with sads-cov were euthanized at 3 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 647 (thermo fisher scientific, usa), anti-cd19-fitc (catalog number 4318813; ebioscience) for b cells, anti-cd4-pe (catalog number 4329629; ebioscience) for t cells, anti-cd11/c-pe-cy7 (catalog number 561022; bd bioscience) for dcs, and anti-f4/80-pe/cy5 (catalog number 123111; biolegend) for macrophages. stained cells were resuspended in 0.2 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 (38) . briefly, each of the plasmids encoding tgev, sars-cov, mers-cov, and mouse hepatitis virus (mhv) s proteins were cotransfected into 293t cells with plenti-luc-green fluorescent protein (gfp) and pspax2 plasmids (kindly provided by zhaohui qian, chinese academy of medical sciences & peking union medical college) at a molar ratio of 1:1:1 by using polyethylenimine (pei). the cells were fed with fresh medium in the next 24 h, and the supernatant media containing pseudovirions were then collected and centrifuged at 800 ϫ g for 5 min to remove debris. to quantify s protein-mediated entry of pseudovirions, mdck cells were seeded at about 25% to 30% confluence in 24-well plates and transfected with either papn-flag, hdpp4-flag, mceacam1a-flag, hace2-gfp (kindly provided by zhaohui qian) (38) , or the control backbone vector by using lipofectamine 3000 (thermo fisher). the mdck cells overexpressing each receptor were inoculated with 500 l of 1:1 diluted corresponding pseudovirions at 24 h posttransfection. at 40 hpi, cells were lysed at room temperature with 110 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, 1) at 24 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 (24) . ethics statement. all animal experiments were performed in strict accordance with the experimental animal ethics committee of zhejiang university (approval number zju20170026). recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission genomic characterization of a newly discovered coronavirus associated with acute 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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-329494-cdn52epy 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: 2009-07-08 journal: antiviral res doi: 10.1016/j.antiviral.2009.07.001 sha: doc_id: 329494 cord_uid: cdn52epy 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 (z1to z4-sirnas) were tested showing variable efficacy. the most effective inhibitor was z2-sirna targeted at the region encompassed by nt 179–197 of z gene. the efficacy of this z2-sirna against junv was also demonstrated in virus-infected cells, by testing infectious virus plaque formation (92.8% junv yield reduction), viral rna level or antigen expression, as well as in cells transfected with z-specific reporter plasmids (91% 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 z2-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 10 to 20% in the absence of the administration of standardized doses of convalescent plasma that is today the best therapy against ahf (damonte and coto, 2002) . 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 g1 and the transmembrane g2, 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, 2001, 2002; lópez et al., 2001) . 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., 2005; perez et al., 2003; salvato et al., 1992; salvato, 1993; strecker et al., 2003) . 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., 1998; campbell-dwyer et al., 2000; djavani et al., 2005) and viral proteins. furthermore, z-mediated budding requires its myristoyl modification (perez et al., 2004) , 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., 2001) . the resulting processed dsrna consists of 21-23 nt fragments with 2-3 nt overhangs at the 3 end of each rna strand (bernstein et al., 2001; zamore et al., 2000) . 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., 2000) . 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., 2001) . 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, 2005) , poliovirus (gitlin et al., 2005) , adenovirus (chung et al., 2007) , human immunodeficiency virus (hannon and rossi, 2004; naito et al., 2007) , hepatitis b virus (li et al., 2007) , hepatitis c virus (liu et al., 2006; lupberger et al., 2008) , influenza a (ma et al., 2007) , marburg virus (fowler et al., 2005) , human papillomavirus (jiang and milner, 2002) , and coronavirus (zhang et al., 2004) , 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., 2005) and lassa virus (lasv) (müller and günther, 2007) . 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-21 cells were grown as monolayers in eagle's minimum essential medium (mem) (gibco) containing 5% fetal bovine serum (gibco) and 50 g/ml gentamycin (sigma). medium was supplemented with 20 m hepes buffer (sigma) when incubated at 37 • c under 5% co 2 . maintenance medium (mm) consisted of mem with 1.5% fetal bovine serum. the attenuated strains iv 4454 and xjcl3 of junv (candurra et al., 1989) , the trlv11573 strain of tacaribe virus (tcrv), and the armstrong 53b strain of lcmv were used. arenavirus stocks were prepared in bhk-21 cells and titrated by plaque formation in vero cells. four double-stranded sirnas were designed targeting the z transcript of junv-iv 4454 (genbank accession no. dq538136) 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 (z1-to z4-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 1) . vero cells grown in six-well plates at approximately 80% confluence were transfected with sirna using lipofectamine 2000 (invitrogen) according to manufacturer's instructions with minimum modifications. briefly, cell supernatant was removed and 500 l opti-mem (gibco) containing 10 l lipofectamine 2000 and 1 m sirna was added. after 4 h of incubation at 37 • c, supernatant was removed and fresh advanced-mem (gibco) containing 2% fetal bovine serum was added to the cells. after 24 h incubation at 37 • c, transfected monolayers were infected with junv at a moi of 0.1. at 24 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 1 h adsorption, cells were washed, covered with mm containing 0.7% methylcellulose, and incubated at 37 • c under 5% co 2 . at day 7 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 (200 u/l, invitrogen) and random primers. this cdna was amplified by real time pcr using sybrgreen (roche) detection. the mix reaction volume was 25 l including 1 l of cdna, dna polymerase gotaq (5 u/l, promega) and specific primers to amplify gene z: z forward 5 -atgggcaactgcaacggggcatc-3 and z reverse 5 -agccaacagcaccaccaccatag-3 . real time pcr was carried out with an initial incubation at 94 • c during 1 min followed by 40 cycles of 30 s at 95 • c, 45 s at 60 • c, 45 s at 72 • c, 2 s at 79 • c and a final incubation of 15 s at 60 • c. amplification plots were expressed as c t values to be analyzed with opticon monitor 3.1. 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 1 m z-sirna and infected with junv at 24 h post-transfection as described in section 2.3. at 24 h p.i., cells were fixed with methanol for 10 min at −20 • c for cytoplasmic immunofluorescence. indirect staining was carried out by incubation with a rabbit anti-junv polyclonal serum for 1 h at 37 • 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 2.5% 1,4-diazabicyclo[2,2,2]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 (5 -ggatccatgggcaactgcaacggggcatc-3 ) and z-trunc-r (5 -aaaagcggccgcctggtggtggtgctgttggct-3 ) 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 pcdna3.1 to generate pcdna3.1-z trunc. oligonucleotides egfp-f (5 -aaaagcggccgcatggtgagcaagggcgaggag-3 ) and egfp-r (5 -aaaatctagattacttgtacagctcgtccatgcc-3 ), 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 pcdna3.1-z trunc vector to generate the reporter plasmid pcdna3.1-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 pcdna3.1-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: 5 -ctggctaactagagaacccactgc-3 and 5 -tatgggccccccggcgcccccatcgatcccaccggtccccatggtttc-ggaggccg-3 . the n and gpc genes were obtained by pcramplification from junv-infected vero cells using the following primers: n agei sense (5 -aaaaccggtatggcacactccaaagagg-3 ), n clai antisense (5 -aaaatcgatcagtgcataggctgcc-3 ), gpc agei sense (5 -gggaccggtatggggcaattcatcagc) and gpc clai antisense (5 -tttatcgatgtgtcctcttgcgcc-3 ). 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 80% confluence on 12 mm diameter glass coverslips were co-transfected with 1 g plasmid pcdna3.1-egfp, pcdna3.1-z-egfp, pcdna3.1-z-flag, pcdnahismax a-n or pcdnahismax a-gpc and 1 m z-sirnas using lipofectamine 2000 (invitrogen) as described previously. as control, cells were transfected with the respective plasmid without z-sirna. to record egfp expression, at 48 h post-transfection cells were fixed with methanol for 10 min at −20 • c, mounted in glycerol with dabco and observed in a fluorescence microscope. for z-flag detection, cells fixed at 48 h post-transfection were incubated with rabbit anti-flag polyclonal serum (cell signaling technology) overnight at 4 • c, followed by incubation with rhodamine-conjugated goat anti-rabbit igg (sigma) for 1 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 sa02bg12 or qc03-bf11 (sánchez et al., 1989) , respectively, then with fitc-labeled goat anti-mouse igg. vero cells co-transfected with 1 g pcdna 3.1-z-flag with or without 1 m z-sirna as described above were harvested at 48 h post-transfection in sample buffer (5% sds, 2% 2-mercaptoetanol, 10% glycerol and 0.005% bromophenol blue in 0.0625 m tris-hcl, ph 6.8) (promega). after boiling the lysates during 7 min, proteins were separated by 15% sds-page and blotted onto a pdvf membrane (millipore). membranes were incubated with a rabbit anti-flag serum overnight at 4 • c. after washing with tris-buffered saline (tbs, 20 mm tris-hcl, 150 mm nacl, ph 7.5) containing 0.1% tween 20, a second incubation was performed with horseradish peroxidase-conjugated anti-rabbit igg during 1 h at room temperature. as control, the presence of actin was revealed by incubation with mouse monoclonal antiactin antibody (jla20, 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 (z1-sirna, z2-sirna, z3-sirna, z4-sirna or x-sirna), and 24 h later cultures were infected with junv at a moi of 0.1. as a control, nontransfected junv-infected cells were also included. supernatants were collected at 24 h p.i. and titrated by plaque assay. cells transfected with the non-specific x-sirnas did not exhibit a reduction in virus yield (1.26 × 10 6 pfu/ml), showing similar levels to nontransfected junv-infected vero cells (1.17 × 10 6 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 24 h incubation period. by contrast, reduction in virus titer was observed in cells treated with the sirnas corresponding to the z transcript (fig. 1a) . whereas z1-sirna reduced the virus titer by 85.3% compared to the viral control, z2-sirna induced a 92.8% yield reduction. in vero cells treated with both z1-sirna and z2-sirna, the virus yield was reduced by 83.7%, showing that the combination of different silencing agents did not present any advantage (data not shown). on the other hand, z3-sirna and z4-sirna were less effective, with only 49.5 and 56.5% of virus yield inhibition, respectively. interestingly, the silencing effect of z2-sirna, the most effective inhibitor, was observed also when the virus yield inhibition was determined at longer period of incubation (48 h p.i.) with 70.5% of virus yield inhibition (fig. 1b) , and even when junv infection was performed at higher mois of 1 and 10, with 77.3 and 75.6% of virus yield inhibition, respectively (fig. 1c) . since the gene regions targeted for z-sirna design were conserved among arenaviruses (fig. 2) , we looked for a possible interference of the selected z-sirnas on xjcl3, 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 4454 were evaluated against junv-xjcl3, a moderate virus yield inhibition was observed with z1-to z4-sirnas, with 43.5, 54.7, 47.7 and 36.6% of virus yield inhibition, respectively. however, when junv z-sirnas were evaluated against the other two arenaviruses species, no inhibition was observed ( fig. 1a 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 z1-sirna and z2-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. 1d) . again, z2-sirna was the most effective inhibitor with a 12-fold reduction in viral rna whereas z1-sirna induced approximately a 3-fold rna inhibition with respect to control x-sirna (fig. 1d) . vero cells treated with z2-sirna and infected with junv were also examined for viral antigen expression at 24 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 g1. in accordance with the reduction in virus yield produced by z2-sirna shown in fig. 1a , a concomitant the xjcl3 (dark grey bars) strains of junv, or tcrv (black bars). supernatants collected at 24 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 z2-sirna and infected with junv, moi = 0.1. supernatants were collected at 24 or 48 h p.i. and titrated by plaque assay. inhibition of virus yield was calculated as above. (c) vero cells were transfected with z2-sirna and infected with junv at different mois. supernatants were collected at 24 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 z1-sirna or z2-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 z2-sirna or x-sirna and infected with junv was detected by immunofluorescence assay using a rabbit anti-junv polyclonal serum. magnification = 400×. decrease in cytoplasmic junv antigen expression was observed (fig. 1e) . 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 pcdna3.1-z-egfp and either the z-sirna or the non-silencing x-sirna. a series of co-transfections of the reporter plasmid pcdna3.1-egfp and the sirnas were assayed in parallel. at 48 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. 3a, panels a and b) . the control non-silencing x-sirnas had no apparent effect on z-egfp expression (fig. 3a, panel c) , whereas the four specific z-sirnas produced a marked reduction in fluorescence (fig. 3a , panels d-g). as observed in virus yield assay, z2-sirna appeared to silence z-egfp gene expression more efficiently than the other z-sirnas. in fact, the quantification of fluorescent cells showed that z1-to z4-sirna reduced expression of the z-egfp fusion protein in comparison to x-sirna by 67.4, 91.0, 46.9 and 45.2%, respectively (fig. 3b) . the specificity of z2-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 3.1-z-flag, by immunofluorescence and western blot assays performed with rabbit anti-flag antibodies. it was evident from the results shown in fig. 3c 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. 45 kda), remained almost unaffected (fig. 3d) . to gain further evidence for the specificity of z-sirnas, we investigated whether the presence of z2-sirna could decrease other transiently expressed junv proteins. to this end, cells were co-transfected with the z2-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. 4 , 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. 1 . 4 . 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 z2-sirna (panels c and f). at 48 h post-transfection, cells were fixed, and protein expression was detected using specific monoclonal antibodies against gpc/g1 and n, followed by fitc-conjugated goat anti-mouse igg (magnification = 400×). 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 z2-sirna, targeted to the region encompassed by nt 179-197 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 z2-sirna on z transcript. interestingly, the inhibitory effect observed in the release of junv infectious progeny by z2-sirna (>90% inhibition at 24 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, 2007) . 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, 2009) . 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., 2003 (garcía et al., , 2006 . 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., 2008) . 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, 2007) , 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 z1-and z2-sirna against iv 4454 and xjcl3 junv strains (figs. 1a and 2). thus, given the variability observed among natural and experimentally isolated junv strains (as shown in fig. 2a for the z gene of iv 4454 , rumero, xjcl3, xj13 and candid 1 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 4e and selectively represses translation in a ring-dependent manner antigenic relationships between attenuated and pathogenic strains of junin virus silencing e1a mrna by rna interference inhibits adenovirus replication characterization of the arenavirus ring finger z protein regions required for z-mediated inhibition of viral rna synthesis ring finger z protein of lymphocytic choriomeningitis virus (lcmv) inhibits transcription and rna replication of an lcmv s-segment minigenome treatment of arenavirus infections: from basic studies to the challenge of antiviral therapy the first targeted delivery of sirna in humans via a selfassembling, cyclodextrin polymer-based nanoparticle: from concept to clinic the proline-rich homeodomain (prh/hex) protein is down-regulated in liver during infection with lymphocytic choriomeningitis virus functional anatomy of sirna for mediating efficient rnai in drosophila melanogaster embryo lysate inhibition of marburg virus protein expression and viral release by rna interference differential inhibitory action of two azoic compounds against arenaviruses arenavirus z protein as an antiviral target: virus inactivation and protein oligomerization by zinc finger-reactive compounds poliovirus escape from rna interference: short interfering 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-7 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 hepg2.2.15 cells rna interference effectively inhibits mrna accumulation and protein expression of hepatitis c virus core and e2 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-1 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 11 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 21 to 23 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-295559-yc8q62z8 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: 2013-10-03 journal: plos one doi: 10.1371/journal.pone.0076469 sha: doc_id: 295559 cord_uid: yc8q62z8 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 s2 domain of group c mers-cov spike protein. mers pseudovirions released from 293t cells contained only uncleaved s, and pseudovirus entry was blocked by lysosomotropic reagents nh(4)cl and bafilomycin and inhibitors of cathepsin l. however, when mers pseudovirions with uncleaved s protein were adsorbed at 4°c to vero e6 cells, brief trypsin treatment at neutral ph triggered virus entry at the plasma membrane and syncytia formation. when 293t cells producing mers pseudotypes co-expressed serine proteases tmprss-2 or -4, 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 2002-03 a previously unknown coronavirus emerged from a wild animal reservoir to cause the sars pandemic, with about 8,000 human cases and more than 770 deaths [1, 2] . previously, cross-species transmission from coronaviruses of bat and bovine origin had allowed human respiratory coronaviruses oc43, nl63 and 229e to become established in the human population worldwide [3] [4] [5] [6] [7] [8] . in the arabian peninsula in 2012, another novel human cov, now called middle east respiratory syndrome coronavirus (mers-cov), was isolated in vero e6 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 70 patients with severe to moderate respiratory disease, 39 of whom have died [9, 10] . 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-hku4 from a tylonycteris pachypus bat and btcov-hku5 from a pipistrellus abramus bat [11] , and to partial sequences of a group c betacoronavirus from a pipistrellus pipistrellus bat in the netherlands [12] . recently group c betacoronaviruses were also detected in a nyctinomops laticaudatus bat in mexico [13] , and nycyteris cf. gambiensis bats in ghana [14] . 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 [13, 15, 16] . the ~200 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 [17] , which typically require protease cleavage between the s1 and s2 domains ( figure 1a ) to permit conformational changes in s2, activated by receptor binding and/or low ph, that mediate membrane fusion leading to virus entry and syncytia formation [3, 17, 18] . 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-2 (tmprss-2), tmprss-4, or cathepsins [18] [19] [20] [21] [22] . 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 1a (mceacam1a) for mouse hepatitis virus (mhv) [23] , a betacoronavirus in group a; aminopeptidase n (apn) for human coronavirus 229e (hcov-229e) and several other alphacoronaviruses [24, 25] ; and angiotensin-converting enzyme 2 (ace2) for both sars-cov, a betacoronavirus in group b and hcov-nl63, an alphacoronavirus [26, 27] . raj and co-workers [28] recently demonstrated that mers-cov uses dipeptidyl peptidase 4 (dpp4) as a receptor. in contrast, s proteins of several group a betacoronaviruses including bovine coronavirus and hcov-oc43 use sialic acid moieties as receptors [29, 58] . 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 293t 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 [30] [31] [32] . codonoptimized cdna encoding s of mers-cov (derived from genbank: afs88936) [15] , with the 16 c-terminal amino acids replaced by a linker, ggggs, and a flag tag (here called mers-cov sδ16) ( figure 1a ) was expressed on 293t cell figure 1b, lane 2) . no protease cleavage products of the ~200kda s protein were detected in transfected 293t cells or pseudovirions ( figure 1b) . in marked contrast, the mers lentivirus pseudovirions used to identify cells susceptible to entry of mers-cov in the poehlmann laboratory [33] , contained a high proportion of cleaved mers-cov s protein at about 100 kda. this important difference in the mers pseudovirions is likely due to differences between our 293t cells and those used in the poehlmann laboratory. surprisingly, when these mers pseudovirions and cell lysates were blotted with polyclonal goat antibody ao4 to spikes purified from detergent-disrupted virions of mhv-a59, a betacoronavirus in group a, the mers s protein bands were detected ( figure 1b ). immunoblotting of soluble, truncated mers s proteins with c-terminal flag tags showed that the ao4 antibody did not recognize the s1 domain of mers s ( figure 1c ), so the cross-reactivity between these proteins from betacoronavirus groups a and c must lie within the s2 domain. vero e6 and llcmk2 monkey kidney cell lines are susceptible to infection with mers-cov virus and to sars-cov [10, 34] , and also susceptible to sars pseudovirions and to mers pseudovirions with uncleaved s protein ( figure 2a ). 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 100 to 1,000 fold increase in luciferase activity in vero e6 and llcmk2 cells (figure 2a and b), and sars pseudovirions showed a 1,000 increase in luciferase activity in vero e6 cells. because the uncleaved mers-cov s protein mediated virus entry into vero e6 and llcmk2 cells, transduction by mers pseudovirions was used to identify additional cell lines that express functional receptors for mers-cov [10, 34] . mers pseudovirions detected strong mers-cov receptor activity on the calu3 line of human airway epithelial cells (figure 2a and b) , and weaker receptor activity on the a549 line of human alveolar basal epithelial cells ( figure 2a ) as also shown by mers-cov infection [35] . interestingly, the eff embryo cell line from eptesicus fuscus bats was susceptible to mers pseudovirions, increasing luciferase activity by nearly 100-fold compared to the no spike control, but the tb1lu lung cell line from tadarida brasiliensis bats, murine fibroblasts and hela cells were not susceptible to mers pseudovirions ( figure 2c ). expression of human ace2 in 293t cells did not significantly increase susceptibility to mers pseudovirions ( figure 3a ), although as expected hace2 greatly increased susceptibility of 293t cells to sars pseudovirions ( figure 3a ). figure 3b and c show that neither human ceacam1, 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 [33] or related human membrane proteins. instead dpp4 is the principal receptor protein for mers-cov [28] . mers pseudovirions induced a small but consistent 5 to 10-fold increase in luciferase activity in 293t human embryo kidney cells compared to the no spike control virus ( figure 2c ), suggesting that our 293t cells expressed either a low level of dpp4, or an alternative but less efficient receptor, such as cd209l or lsectin for sars-cov [36, 37] . 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 e6 cells, 20mm nh 4 cl inhibited entry of sars pseudovirions by about 99.9% compared to entry of sars pseudovirions without inhibitor, and nh 4 cl also inhibited entry of mers pseudovirions by about 99.6% ( figure 3a ). bafilomycin a specifically inhibits the vacuolar-type h+-atpase that is required for acidification of lysosomes. figure 3a shows that bafilomycin a inhibited entry of sars pseudovirions into vero e6 cells by 99.8% as previously reported [21, 38] , and also inhibited entry of mers pseudovirions by more than 99.9% compared to mers pseudovirions without inhibitor. in llcmk2 cells, although bafilomycin a inhibited 99.7% of mers-cov s mediated entry, nh 4 cl reduced mers-cov s-mediated entry only 6-fold (data not shown), suggesting that the inhibition of endosomal acidification by nh 4 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 [39] , sars-cov [22] , and ebolavirus [40] . e64d, an inhibitor of the cysteine protease activities of cathepsins b, h, and l and calpain, reduced transduction of vero e6 cells by sars pseudovirions by 80% as previously reported ( figure 3b ) [41] . since cell entry mediated by vsv-g glycoprotein does not require protease activation [17] , e64d treatment of vero e6 ( figure 3b ) and llcmk2 cells (data not shown) did not inhibit entry of vsv pseudovirions. however, e64d decreased entry into vero e6 cells of mers pseudovirions with uncleaved s by 96.7% ( figure 3b ) and llcmk2 cells by 99.2% (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 [41] , in vero e6 cells 10 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 3b ). cathepsin l inhibitor iii reduced entry into vero e6 cells of mers pseudovirions with uncleaved s protein by 97% relative to entry without inhibitor ( figure 3b ), and similar results were seen in llcmk2 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 e6 and llcmk2 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 4°c to ace2 on the cell membrane are treated with trypsin, then warmed to 37°c in the presence of an inhibitor of endosomal acidification [21] . trypsin treatment at either 4°c or 37°c cleaved the s protein of mers pseudovirions and generated a ~65kda subunit in the s2 domain of the protein recognized by antibody to mhv-a59 s protein ( figure s1 ). mers pseudovirions with uncleaved s protein were adsorbed at 4°c to cell surface receptors on vero e6 cells in the presence of 20mm nh 4 cl, and then the cells with bound virions were briefly treated with trypsin at ph 7.4 at room temperature to cleave the ~200 kda s protein and activate its membrane fusing activity. figures 3a and 4a show that nh 4 cl strongly inhibited infection of vero e6 cells by mers pseudovirions with uncleaved s. however, trypsin treatment of the mers pseudovirions bound at neutral ph and 4°c to the vero e6 cell membrane triggered both virus entry at the plasma membrane and formation of small syncytia by 40 hours post inoculation ( figure 4a 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. 293t cells expressing uncleaved mers-cov s protein or control cells stably transfected with the empty pcdna3.1 vector were overlaid on monolayers of vero e6 cells in the presence or absence of tpck trypsin ( figure 4c ). no syncytia formation was induced by 293t cells with empty vector or 293t cells expressing mers-cov sδ16 without trypsin ( figure 4c ), but addition of tpck trypsin to the medium triggered formation of massive syncytia in the vero e6 cells co-cultured for 20 hr with mers-cov s-expressing 293t cells ( figure 4c , arrows). large syncytia were also formed after even a brief 20 minute trypsin pre-treatment at ph 7.4 and 4°c of 293t cells expressing mers-cov s protein, followed by incubation with a 5-fold excess of soybean trypsin inhibitor before layering the cells over confluent monolayers of vero e6 cells and incubating at 37°c for 20 hours ( figure 4c, lower central panel, arrows) . thus, trypsin cleavage at neutral ph of mers-cov s protein on 293t cells triggered syncytia formation when s was bound to receptors on susceptible vero e6 cells. type ii transmembrane serine proteases, including tmprss-2 and tmprss-4, 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 [19, 20, [42] [43] [44] [45] . we therefore transfected 293t cells with plasmids encoding tmprss-2 or -4, mers-cov sδ16 protein, pspax2 and plenti-gfp-luc and investigated whether s proteins on pseudovirions produced in these cells were cleaved and whether they could infect vero e6 cells in the presence of nh 4 cl. surprisingly, the pseudovirionproducing 293t cells expressing either tmprss-2 or -4, formed large syncytia by 40 hrs after transfection ( figure 5a ), but the mers pseudovirions produced by these cells could not transduce vero e6 cells in the presence or absence of nh 4 cl ( figure 5b ). in contrast, without tmprss-2 or -4, the 293t cells expressing uncleaved mers-cov sδ16 did not form syncytia, and pseudovirions that they produced efficiently infected vero e6 cells, but virus entry was inhibited by nh 4 cl ( figure 5b ). immunoblots with antibody ao4 to mhv s or anti-flag (data not shown) revealed that the mers pseudovirions produced in 293t cells expressing tmprss-2 or -4 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 [10, 16, 34] , 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 2. we found that mers pseudovirions, like infectious mers-cov virions [10, 34] , readily infected the vero e6 and llcmk2 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 [10, 33, 35, 46] . muller et al. [34] 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 [47] , including a diverse array of both old world and new world bats, suggests that the receptor for mers-cov, dpp4 [28] , is broadly conserved among many species, an important property of many emerging viruses [47, 48] . e. fuscus figure 5a were inoculated onto vero e6 cells in the presence (striped bars) or absence (white bars) of 20mm nh 4 cl to inhibit acidification of endosomes. doi: 10.1371/journal.pone.0076469.g005 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 [49, 50] . 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 96% similarity to mers-cov [13] , coupled with the diverse array of alphacoronaviruses previously discovered in north american bats [49] [50] [51] [52] , 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-a59, a group a betacoronavirus. the cross-reactive epitope(s) was mapped to the s2 domain, which is more highly conserved than the s1 domain of betacoronaviruses. chan et al. [33, 53] 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 s2 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 [17] . 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 gp120, low ph in endosomes like influenza ha, or both like avian sarcoma leukosis virus (aslv) [9, 17, [54] [55] [56] . coronaviruses in different phylogenetic groups differ in the sequence of steps leading to virus entry [57, 58] . the s protein on virions of group a betacoronavirus mhv-a59 requires protease activation--either by furin during virus maturation [18] , 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 [59] . in contrast, virions of group b betacoronavirus sars-cov that contain uncleaved s [22] , first bind to the viral receptor protein, ace2, 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 [21, 22, 60] . 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 293t cells contained only uncleaved s protein, and, as for most coronaviruses, cleavage between s1 and s2 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 [33] reached similar conclusions using mers pseudovirions that, unlike ours, contained more cleaved than uncleaved s protein, although both labs had made the pseudovirions in 293t cells. gierer et al. [33] showed that batches of 293t cells differ markedly in expression of the mers-cov receptor and susceptibility to transduction by mers pseudovirions. in our laboratory, the 293t 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 [21, 22] , mers pseudovirions could enter susceptible vero e6 cells at the plasma membrane if virions were first bound to cell surface receptors at 4°c at neutral ph in the presence of nh 4 cl to inhibit acidification of endosomes, and also treated briefly at room temperature with trypsin to cleave the viral s protein. upon warming to 37°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 [33] 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 s1/s2 heterodimer dissociates before receptor binding. we found that mers pseudovirions released from cells expressing tmprss-2 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 [33, 53] , could not enter cells at the plasma membrane because s1 may have dissociated from the cleaved spikes on virions. development of antibodies specific for the s1 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-3 and caco-2 cell lines induced syncytia formation [35] . our 293t cells were only minimally susceptible to entry of mers-pseudovirions, and did not form syncytia when producing mers pseudovirions with uncleaved s. however, when 293t cells expressing mers-cov sδ16 protein were co-cultured in the presence of trypsin with vero e6 cells that express the mers-cov receptor, enormous syncytia formed. these observations suggest that in tissues such as the lung, where trypsin, tmprss-2 or -4 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 [61, 62] . when s proteins expressed on the plasma membrane are cleaved, the s1 domain can detach from the spike, exposing the hydrophobic fusion peptide of the membrane-anchored s2 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 s1/s2 interactions, and low stability of s1/s2 heterodimers correlates with rapid spread of infection through tissues that express little receptor protein [63, 64] . we found that transmembrane serine proteases tmprss-2 and -4 could activate the syncytia forming activity of mers-cov sδ16 protein expressed in 293t cells as these proteases do for class 1 fusion proteins of other respiratory viruses including influenza, sars-cov and human metapneumovirus [19, 20, [42] [43] [44] [45] . we were surprised that 293t 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 [33, 53] . most neutralizing antibodies would likely target the receptorbinding s1 domain of mers-cov s, which is less conserved than the s2 domain and can mutate, likely generating antibody escape mutants [65] . here we showed that a polyclonal antibody to the s protein of mhv, a group a betacoronavirus, cross reacts with the s2 domain of mers s protein in immunoblots. as we and others have proposed for sars-covs [66, 67] , the more highly conserved s2 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, tmprss2 or -4 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 [15] was synthesized with the c-terminal 16 amino acids replaced with a ggggs linker and a flag tag (genscript, piscataway, nj), and for eukaryotic expression was cloned into pcdna3.1(+) (invitrogen) between the bamhi and noti sites. to make constructs for expression of truncated soluble mers s aa1-351, s aa1-384, and s aa1-748 proteins, pcr reactions were performed using the same forward primer aatgaaaagcttcaccatgattcactccgtgttcctc pairing with the following reverse primers, for s aa1-351: tagttttctagaacttccgcctccaccataa ctacagtggagctggct; for s aa1-384: tagttttctagaacttccgcctccac cgtcgcactccacgccttctgcc; or for s aa1-748 tagttttctagaacttc cgcctccacctggggtcagtgtgctgggggt, and cloned into p3xflag-cmv 14 (sigma, st louis, mo) between hindiii and xbai sites for expression. the vsv-g plasmid and lentiviral packaging plasmid, pspax2, 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 [68] the vero e6 line of african green monkey kidney cells, the 293t line of human embryonic kidney cells transformed with sv40 large t antigen, the calu 3 line of human airway epithelial cells, the a549 line of human alveolar epithelial cells, and the tb1lu 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 [69] . 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 03-096a from the colorado state university iacuc. murine nih3t3 cells stably expressing recombinant human aminopeptidase n (hapn) or control cells with empty vector were previously described [70] . these cell lines were maintained in dulbecco's mem with 10% fetal bovine serum (fbs) and 2% penicillin, streptomycin, and fugizone (psf) (life technologies inc, grand island, ny). the llc-mk2 line of rhesus monkey kidney cells from atcc ccl-7 was maintained in opti-mem1 (life technologies inc, grand island, ny) with 10% fbs and 2% psf. pseudotyped lentiviruses were produced as described previously [71] with minor modifications. briefly, plasmids that encode viral spike glycoproteins mers-cov s∆16, sars s∆19, or vsv-g were co-transfected into 293t cells with pspax2 and plenti-gfp-luc using polyetherimide (pei) (polyscience inc, warrington, pa). forty to 60 hr later the supernatant media containing pseudovirions were centrifuged at 800g for 5min to remove debris, and passed through a 0.45µm filter. to quantitate entry of pseudovirions into different cell types, 250µl of pseudovirions with 8 µg/ml of polybrene (sigma) was inoculated onto cells in 24-well plates, incubated overnight at 37°c, and cells were fed with fresh medium. at 40hr post inoculation (pi) cells were lysed at room temperature with 120µl 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 sδ16, sars sδ19, or vsvg glycoprotein or control pseudovirions with no spike were centrifuged through a 20% sucrose cushion at 30,000 rpm at 4°c for 3 h in a beckman sw41 rotor [71] . spike proteins in the virions were separated on 4-15% sds page, blotted to nitrocellulose and detected with mouse anti-flag antibody m2 (sigma, st louis, mo) for mers-cov sδ16, or polyclonal goat antibody ao4 to purified spikes from detergent-disrupted mhv-59 virions [72] , followed by horseradish peroxidase (hrp)-conjugated antibody to mouse or goat igg, and visualized with chemiluminescent reagent plus (perkinelmer, boston, ma). vero e6 cells or llcmk2 cells were incubated for 1hr at 37°c with medium alone or medium containing either 20mm nh 4 cl or 200nm bafilomycin a to inhibit acidification of endosomes, and then spin-inoculated with pseudovirions and no spike controls in the presence of either 20mm nh 4 cl or 200nm bafilomycin a for 90 min at 1,200g at 4°c. at 40 hr pi cells were lysed and luciferase activity was quantitated as a measure of virus entry. in figure 3 , the luciferase activities of vero e6 cells transduced with vsv-, sars-, and mers-covspseudovirions without endocytosis inhibitor were 1x10 6 , 1.5-2x10 6 , and 5-6x10 5 , respectively. monolayers of vero e6 or llcmk2 cells were incubated with 20mm nh 4 cl in medium containing 10% fbs for 1hr at 37°c, then shifted for 15min to 4°c with 40mm nh 4 cl. pseudovirions were adsorbed to cells at 4°c by spin-inoculation for 90 min at 1,200g. the virus inocula were removed and replaced with prewarmed, serum-free dmem with or without 20mm nh 4 cl. after 15min at 37°c, the media were replaced with serum-free dmem with or without 15 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 15 min on ice with 75µg/ml of soybean trypsin inhibitor (worthington biochemical corporation, lakewood, nj) in dmem with 10% fbs, and cells were incubated overnight at 37°c, fed with fresh medium, and incubated at 37°c. at 40 hr pi, virus entry was quantitated by luciferase activity and cells were photographed to detect viral cytopathic effects. monolayers of vero e6 cells or llcmk2 cells were preincubated for 2 hrs at 37°c with either 30µm of e64d, that inhibits cathepsin b, h, l and calpain, or 10µm 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 4°c, then incubated at 37°c for 5 hours with or without the endocytosis inhibitors. cells were then incubated at 37°c without inhibitors, and at 40 hr pi, luciferase activity in lysed cells was determined. 293t cells in 6-well plates were transfected using pei with 3µg of either empty vector or mers-cov s∆16 plasmid. for brief trypsin pre-treatment, cells were lifted with 1mm edta in pbs and washed, then incubated on ice with 20 µg/ml of tpck trypsin or control medium for 20 min. typsin activity was then inhibited by incubation with a five-fold excess of soybean trypsin inhibitor for 15min. the trypsin-pretreated 293t cells and control cells were then layered over monolayers of vero e6 cells and incubated for 20 hr. syncytia formation was detected by phase contrast microscopy or by imaging fixed cells stained with crystal violet. figure s1 . 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 20 µg/ml of trypsin either at 4°c or 37°c for 20 min. after digestion, glycoproteins on pseudovirions were analyzed by immunoblot using ao4 antibody to the s protein of murine betacoronavirus mhv-a59 that cross-reacts with mers-cov s protein. lanes 1 to 3: no spike control; lanes 4 to 6: mers pseudovirions; lanes 1 and 4: no trypsin; lanes 2 and 5: trypsin at 4°c; lane 3 and 5: trypsin at 37°c. the band at ~90kda was seen in pseudovirions without spike, indicating that it is not due to mers-cov s protein. uncleaved mers-cov s protein, 200kda, on pseudovirions is shown in lane 4, and trypsin treatment cleaved all of the s protein, and generated a subunit of ~65kda that was recognized by ao4 antibody (lanes 5 and 6). 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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 cd66 carcinoembryonic antigens mediate interactions between opaexpressing neisseria gonorrhoeae and human polymorphonuclear phagocytes human myeloid plasma membrane glycoprotein cd13 (gp150) 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 pca7-tmprss2 and pcmv-tmprss4 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-289248-6mx4o0eb 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 hu191 measles virus vaccine by alteration of the s-adenosylmethionine (sam) binding site in the large polymerase protein date: 2018-05-01 journal: virology doi: 10.1016/j.virol.2018.02.022 sha: doc_id: 289248 cord_uid: 6mx4o0eb the live-attenuated measles virus (mv) vaccine based on the hu191 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-adenosylmethionine (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-hu191 strain and generated two recombinant mv-hu191 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-hu191 vaccine strain. importantly, both mv-hu191 mutants triggered a higher neutralizing antibody than rmv-hu191 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, 2009; tangy and naim, 2005) , though vaccination is an effective, economical, and safe way to prevent outbreaks (bester, 2016; de vries et al., 2008) . in early 1960, a live-attenuated vaccine based on the hu191 strain of mv was developed and is currently widely used for immunization in all provinces of china (zhang et al., 2009) . 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, 2016; shu et al., 2011) . 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., 2016; zhang et al., 2016) . 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, 2015; gao et al., 2017; ma et al., 2016; zhang et al., 2016) . 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., 2002) . 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., 1996; clarke et al., 2000; garcin et al., 1995; gassen et al., 2000; jin et al., 1998; lawson et al., 1995) . 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., 1999; kovacs et al., 2003; nakatsu et al., 2006; parks et al., 1999; radecke et al., 1995; sidhu et al., 1995) . 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., 2002; poch et al., 1990; whelan et al., 2004) . during transcription, nns rna viruses synthesize mrnas that are capped and methylated at the 5'end and polyadenylated at the 3' 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., 2002; furuichi and shatkin, 2000; ogino and banerjee, 2007; zhang et al., 2014) . 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., 2008; ogino and banerjee, 2007) . the mrna cap methylation in nns rna viruses is also unusual in that a single region in the l protein catalyzes both guanine-n-7 (g-n-7) and ribose 2′-o (2′-o) methylation (li et al., 2006; rahmeh et al., 2009) . 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., 2008; ogino et al., 2005) . 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., 2006; sun et al., 2014; tian et al., 2015; zhang et al., 2014) . 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-191, allowing us to recover recombinant mv in bhk cells stably expressing t7 rna polymerase (xu et al., 2011; zhang et al., 2014) . subsequently, two recombinant mvs with amino acid (aa) substitutions in the s-adenosylmethionine (sam) binding site of l protein (rmv-hu191-g1788a and rmv-hu191-g1792a) were successfully recovered. these two mtase-defective mutants had delayed replication kinetics, grew to high titers, and were genetically stable through 15 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. 1 . construction of a full-length cdna clone for mv-hu191. the t7 promoter, 3′ and 5′ non-coding termini (nct), antigenomic hdv ribozyme and t7 terminator were assembled in several rounds of fusion pcr, and inserted into pyes-2 using a "seamless" cloning strategy, resulted in the construction of p107109-mv(+) (a). eight overlapping fragments containing the full-length mv genome were assembled into p107109-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 hu191, pyes-mv(+), was constructed by a novel methodology using the geneart™ high-order genetic assembly system. the full-length cdna clone of mv-hu191 was successfully assembled by a single step ligation, without the need for restriction endonucleases. the 15,896-nt antigenomic mv cdna was cloned under the control of a t7 rna polymerase promoter, a hepatitis delta virus (hdv) ribozyme sequence, and a t7 terminator ( fig. 1 ; table 1 ). to recover infectious mv, bhk-sr19-t7 cells stably expressing t7 rna polymerase were co-transfected with full-length cdna clone pyes-mv(+) and the support plasmids expressing ribonucleoprotein (pt7-hu191-n, pt7-hu191-p, and pt7-hu191-l). three days post-transfection, cell monolayers were trypsinized and co-cultured with fresh vero cells. mv-induced syncytia were observed 2-3 days later ( fig. 2a , b and c). the successful recovery of rmv-hu191 was further confirmed by detection of n protein expression in vero cells infected with the rescued rmv-hu191 by an immunofluorescence assay (fig. 2d ). when extensive syncytia were observed, cells were harvested and the supernatants were used for further passage in vero cells. after 3-4 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 8763 within the h gene of the lab-propagated parental virus, which is different from the published hu191 sequence (genbank accession no. fj416067). 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-hu191) originated from pyes-mv(+) and not from cross-contamination of the parental mv-hu191 grown in our laboratory, a region of the rmv h gene was amplified by rt-pcr and sequenced. the result showed that rmv-hu191 contained the "c" mutation. having the establishment of robust reverse genetics for rmv-hu191, we next tested the hypothesis that the rmv-hu191 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., 2006; sun et al., 2014; zhang et al., 2014) . 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., 1997; schluckebier et al., 1995) . 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. 3) (li et al., 2006; mcilhatton et al., 1997; poch et al., 1990; zhang et al., 2014) . sequence analysis found that aa residues corresponding to the gxgxgx motif of the mv l protein includes g1788, g1790, and g1792. 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 (g1788a, g1790a or g1792a) in their sam binding sites were constructed. using the reverse genetics system, two recombinant mvs, rmv-hu191-g1788a and rmv-hu191-g1792a, 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. 2e and 2f). the rmv-hu191-g1790a 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-hu191-g1788a and rmv-hu191-g1792a were plaque purified. the sizes of virus-induced plaques differed between the rescued parental and mutant viruses. as demonstrated in fig. 4 , after 6 days of incubation, the parental rmv-hu191 formed plaques were 1.43 ± 0.22 mm in diameter, whereas the average plaque for rmv-hu191-g1788a and rmv-hu191-g1792a was significantly smaller (0.98 ± 0.16 mm and 0.79 ± 0.13 mm, respectively; p < 0.05). 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-hu191 mutants and the parental virus in vero cells in the time course of 120 h after infection (fig. 5 ). parental rmv-hu191 reached a peak titer (6.86 ± 0.14 log 10 pfu/ml) at 72 h post-inoculation (hpi), while peak titers for the two mutants at 96 hpi. importantly, rmv-hu191-g1788a was delayed in replication but reached a peak titer of 6.9 ± 0.06 log 10 pfu/ml at 96 hpi, which was comparable to the parental virus (p > 0.05). the peak titer achieved by rmv-hu191-g1792a was 6.06 ± 0.20 log 10 pfu/ml at 96 hpi, which was significantly lower than that of parental rmv-hu191 at 72 hpi (p < 0.05). both mv mutants had delayed cytopathic effects (cpe) compared to the parental virus. the parental rmv-hu191 developed extensive cell-to-cell fusion and large syncytia at 48 hpi and reached maximum cpe at 72 hpi, whereas mutants rmv-hu191-g1788a and rmv-hu191-g1792a had a delay in formation of syncytia and reached maximum cpe at 96 hpi (fig. 6) . 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-hu191-g1788a and rmv-hu191-g1792a were genetically stable in vitro, each virus was passaged in vero cells for 15 times. the mutated region in the l gene was sequenced for each of the first 10 passages. virus in each passage retained the desired mutation. at passage 15, the entire genome of each mutant was sequenced, showing no additional mutations in the genome. table 1 sequences of the oligonucleotides used for pcr. sequence (5′−3′) virology 518 (2018) 210-220 2.5. rmv-hu191 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-hu191 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 4 post-inoculation, cotton rats were terminated, and viral titer in the lungs was determined ( table 2 ). the parental virus replicated efficiently in lungs with an average titer of 3.75 log 10 pfu/g lung tissue. recombinant rmv-hu191-g1788a had an average titer of 3.08 log 10 pfu/g lung tissue, which was significant lower than rmv-hu191 (p < 0.05). however, rmv-hu191-g1792a was the most attenuated mutant. only 1 out of 5 cotton rats had detectable viral titer in the lung with a titer of 2.56 log 10 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-hu191-g1788a or rmv-hu191-g1792a from each animal. no additional mutations were detected in the sequenced region. 2.6. rmv-hu191 mutants induce higher neutralizing antibodies than the parental vaccine strain and provide complete protection against mv challenge the immunogenicity of rmv-hu191 mutants was assessed in cotton rats. briefly, 4-6-week-old spf cotton rats were intranasally inoculated with 1.0 × 10 6 pfu of each mv, and were challenged with 1.0 × 10 7 pfu of rmv-hu191 at week 4 post-immunization. the two mutant rmvs induced high levels of neutralizing antibodies as early as 1 week after vaccination, and antibodies gradually increased from weeks 2-4. however, antibodies produced by the parental rmv-hu191 peaked at week 2, and declined during weeks 3 and 4. overall, the antibodies induced by rmv-hu191-g1788a and rmv-hu191-g1792a were comparable to those generated by wild-type rmv-hu191 at weeks 1-3 (p > 0.05) (fig. 7a) . however, at week 4, neutralizing antibodies induced by rmv-hu191 mutants were significantly higher than those from parental rmv-hu191 (p < 0.05; fig. 7b ). this suggests that rmv-hu191 mutants were more immunogenic compared to the parental vaccine strain. at week 4 post-vaccination, cotton rats were challenged with 1.0 × 10 7 pfu of rmv-hu191 and all cotton rats were terminated at day 4 post-challenge. no infectious virus was detected in the lung tissue of any of the vaccinated cotton rats (table 3 ). in contrast, an average titer of 4.02 ± 0.37 log 10 pfu/g was detected in lung tissue from unvaccinated but challenged controls. these results show that rmv-hu191-g1788a and rmv-hu191-g1790a 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-hu191-g1788a and rmv-hu191-g1792a were significantly more attenuated compared to parental rmv-hu191, 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-hu191-g1788a grew to high titer in vero cells that was comparable to rmv-hu191 but exhibited significantly more attenuation in cotton rats. recombinant rmv-hu191-g1792a grew to a relatively lower titer in vero cells but had a greater degree of attenuation in cotton rats compared to rmv-hu191-g1788a. both recombinant viruses triggered significantly higher neutralizing antibody compared to rmv-hu191, 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-hu191 vaccine strain. we established a more efficient method to assemble a full-length cdna clone of mv-hu191 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-7 or mva-t7 providing t7 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 t7 rna polymerase, instead, were used to rescue hmpv and bovine respiratory syncytial virus (buchholz et al., 1999; zhang et al., 2014) . in our study, bhk-sr19-t7 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 hu191 strain of mv has been developed and is widely used for immunization in chinese infants and children (zhang et al., 2009 ). 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, 2015; gao et al., 2017; ma et al., 2016; zhang et al., 2016) . 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-hu191, rmv-hu191 carrying mutations in the sam binding site, rmv-hu191-g1788a and rmv-hu191-g1792a, were successfully rescued. these rmv-hu191 mutants produced smaller plaques, had delayed growth kinetics, and had delayed syncytia formation compared to parental rmv-hu191. clearly, both mutants were significantly more attenuated in vero cells than the parental rmv-hu191. in cotton rats, rmv-hu191-g1788a had a significantly lower viral titer in the lungs than rmv-hu191 (p < 0.05). recombinant rmv-hu191-g1792a was even more attenuated, as only 1 out of 5 inoculated cotton rats had detectable viral titer in the lung. despite the high attenuation phenotype, rmv-hu191-g1788a grew to high titer compared to parental vaccine virus in vero cells, the who approved cell line for vaccine production. although rmv-hu191-g1792a grew to a slightly lower titer (0.5 log less) in vero cells, it is still economically feasible for vaccine production. another advantage of using rmv-hu191-g1792a is that it had a greater degree of attenuation in vitro and in vivo compared to rmv-hu191-g1788a. interestingly, both mutants triggered a higher level of neutralizing antibodies than parental rmv-hu191, suggesting their greater immunogenicity. finally, cotton rats vaccinated with both mutants were completely protected from the mv challenge. thus, recombinant rmv-hu191 carrying mutations in the sam binding site are potentially improved vaccine candidates for mv. a novel finding is that recombinant rmv-hu191 carrying mutations in the sam binding site triggered a higher neutralizing antibody compared to the parental rmv-hu191 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 2'-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 2′-o methylation but not g-n-7 methylation, and were significantly attenuated but retained wild-type levels of immunogenicity. in addition, it was found that all 2′-o mtase-defective hmpvs were highly sensitive to ifn-α and ifn-β treatment (sun et al., 2014; zhang et al., 2014) . 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 2′-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-hu191 and established an efficient vaccinia virus-free reverse genetics system for mv-hu191. we generated two recombinant mv-hu191 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-81) and bhk-sr19-t7 cells (kindly offered by apath, llc, brooklyn, ny) were grown in dulbecco's modified eagle's medium (dmem; life technologies) supplemented with 10% fetal bovine serum (fbs). the chinese hu191 vaccine strain of mv (obtained from dr. yiyu lu, zhejiang cdc) was passaged in vero cells. viral rna was extracted from 200 µl of mv-hu191 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 q5® high-fidelity 2 × master mix (neb), using eight pairs of mv-specific primers (table 1) , and cloned into the peasy-blunt vector (transgen) according to the manufacturer's instructions. the resultant eight plasmids containing the full-length mv-hu191 genome (peasy-n, peasy-p, peasy-m1, peasy-m2, peasy-f, peasy-h, peasy-l1, peasy-l2) were sequenced, and found to be identical with the published sequence of mv-hu191 (genbank accession no. fj416067), except for a single point change (c to u) at nt 8763 within the h gene. this mutation in peasy-h was corrected by site-directed mutagenesis with specific primers ( table 1 ). the resultant plasmid was named peasy-h-m. several rounds of amplification and "in-fusion" pcr were used to assemble five fragments [the t7 promoter, mv 3′ and 5′ non-coding termini (nct) (3′−107 nt and 5′−109 nt, respectively) were amplified from peasy-n and peasy-l2, respectively, hepatitis delta virus (hdv) ribozyme (84 nt of anti-genomic hdv sequence), and the t7 terminator], and subsequently inserted them into the pyes-2 plasmid using the geneart™ seamless cloning and assembly kit (invitrogen; fig. 1a ), creating plasmid p107109-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. 1b) , creating plasmid pyes-mv(+). eight mv-hu191 genomic fragments were amplified with specific primers from peasy-n, peasy-p, peasy-m1, peasy-m2, peasy-f, peasy-h-m, peasy-l1, and peasy-l2. the p107109-mv(+) insert (containing the t7 promoter, mv 3′ and 5′ ncts, hdv ribozyme, and t7 terminator) was divided into two fragments by pcr amplification with specific primers (f:5′-ttctgccgcctgcttcaaaccg-3', r:5′-ctcggatatccctaatcc-3'; f:5′-ttggttgaactccggaac-3', r: 5′-cagaatgggcagacattacgaatgc-3'). a backbone vector pt7, which contains the t7 rna polymerase promoter, encephalomyocarditis (emc) virus internal ribosome entry site (ires), and the t7 terminator sequences, was used to construct plasmids encoding mv-hu191 n, p, and l genes. the open reading frames (orfs) of the mv hu191 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 hu191 l gene was amplified from peasy-l1 and peasy-l2 using two primer pairs, mv-cds-l1 (+)/(-) and mv-cds-l2 (+)/(-). the mv n, p, and l genes were inserted into the pt7 vector between the ires and polya table 2 replication of rmv-hu191 mutants in cotton rats. % infected animals viral titer (log 10 pfu/g) b,c rmv-hu191 100 3.75 ± 0.12 a rmv-hu191-g1788a 100 3.08 ± 0.33 b rmv-hu191-g1792a 20 2.56 b dmem 0 nd nd: not detected. a each cotton rat was inoculated intranasally with 5.0 × 10 6 pfu of rmv-hu191 or rmv-hu191 mutants in a volume of 100 µl. at day 4 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. 7 . neutralizing antibody titers produced by cotton rats after inoculation with recombinant mv. cotton rats were intranasally inoculated with 1.0 × 10 6 pfu of rmv-hu191 or rmv-hu191 mutants in 0.1 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 < 0.05) of neutralizing antibodies at 4 weeks post-inoculation. * =p < 0.05; * *=p < 0.01; *** =p < 0.001. sequences using a "seamless" cloning strategy, resulted in the construction of pt7-hu191-n, pt7-hu191-p, and pt7-hu191-l, respectively. the primer sequences used in the pcr assays are available upon request. to recover recombinant mv, bhk-sr19-t7 cells were grown overnight in six-well plates to approximately 90% confluence, and were transfected with 5 µg of pyes-mv(+), 1.5 µg of pt7-hu191-n, 1.5 µg of pt7-hu191-p, and 0.5 µg of pt7-hu191-l using previously described procedure (carsillo et al., 2009; kovacs et al., 2003; singh and billeter, 1999) . at 72 h post-transfection, cell monolayers were trypsinized and directly transferred onto vero cell monolayers (p0) at 75% confluence and co-cultured at 37°c for 3-5 days. cells were subjected to three freeze-thaw cycles when extensive cpe (mv-induced syncytia) was observed. after a brief centrifugation, supernatants (p1) were harvested and used for further passages on confluent vero cell monolayers. at p2 or p3, the recovered viruses were plaque purified and sequenced. vero cells grown in 24-well tissue culture plates were infected with rmv or rmv-mutant. after 1 h incubation, the cells were washed two times with pbs before cultivating them in dmem containing 2% fbs. at 24 or 48 h postinfection, the cells were fixed with 4% paraformaldehyde in pbs for 20 min at rt, permeabilized with 0.1% triton x-100 (merck millipore) in pbs for 10 min at rt, and blocked with 1% bsa in pbs containing 0.05% tween 20 for 1 h at rt. the cells were stained with mouse anti-measles virus n antibody (ab106292, abcam) for 1 h at rt. after washing with pbs, alexa fluor ® 594 donkey anti-mouse igg (h+l) (a21203, invitrogen) were added and incubated for 1 h at rt. then, the cells were stained with 4,6-diamidino-2-phenylindole (dapi) for 10 min at rt. images were obtained using a zeiss clsm780 confocal laser scanning microscope and zen 2012 software. amino acids (g1788, g1790, and g1792) in the sam binding site in the hu191 l protein were mutated to alanine individually (ggt to gct at aa posi tion 1788, gga to gca at aa position 1790, and ggt to gct at aa position 1792; the mutated nucleotides were underlined) in an infectious cdna clone of mv-hu191 [pyes-mv(+)] using a q5® sitedirected mutagenesis kit (neb) according to the manufacturer's instructions. the resultant plasmids were named pyes-mv(+)-g1788a, pyes-mv(+)-g1790a, and pyes-mv(+)-g1792a. mutations were confirmed by dna sequencing. the titers of rmv-hu191 viruses were determined by plaque assay in vero cells. briefly, vero cells were seeded in six-well plates at a density of 5 × 10 5 cells per well, incubated for 18 h, and the medium was removed prior to infection of cell monolayers with serial dilutions of rmv-hu191. after 1 h of adsorption with constant shaking, the medium was removed and cell monolayers were covered with 2.5 ml of eagle's minimal essential media (mem) containing 2% agarose, 0.75% sodium bicarbonate(nahco3), 5% fbs, 20 nm hepes, 2 mm l-glutamine, and 4 mg/ml of streptomycin. at 6 dpi, cells were fixed in 4% (vol/vol) paraformaldehyde for 2 h, and the plaques were visualized by staining with 0.05% (wt/vol) crystal violet. confluent vero cells in six-well plates were infected with rmv-hu191 viruses at a multiplicity of infection (moi) of 0.01. after 1 h incubation, the inoculum was removed and the cells were washed three times with pbs. fresh maintenance media (dmem supplemented with 2% fbs) was added, and the infected cells were incubated at 37°c. at different time points post-infection, the cells were harvested by three freeze-thaw cycles, and the supernatant collected by centrifugation at 3000×g in an allegra 6r centrifuge (beckman coulter) for 15 min. virus titers were determined by plaque assay in vero cells. confluent vero cells in t25 flasks were infected with each rmv-hu191 mutant at an moi of 0.01, and cell culture supernatants were collected after appearance of cpe and used to infect new confluent vero cells in fresh t25 flasks. each mutant was serially passaged 15 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 15. 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 1.2 kb dna fragment of the h protein gene was amplified by a one-step rt-pcr kit (qiagen) using primers rmv-h-8583-forward (5'gttcagggatggacctatac-3') and rmv-l-9793-reverse (5'ggtgtgtgtctcctcctat-3'). 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-hu191 grown in our laboratory. a 1.1 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-14128-forward (5'-gaccggtagagaaatgtgcag-3') and rmv-l-15222-reverse (5'-gcttaatggataggatgtgac-3'). pcr products were sequenced to confirm that each recombinant virus contained the desired mutation. the rmv-hu191 stocks for use in animal experiments were grown in vero cells and purified by ultracentrifugation. twenty t150 flasks with confluent vero cells were infected with each rmv at moi of 0.01. after 1 h of adsorption with constant shaking, 15 ml of dmem (supplemented with 2% fbs) was added to each flask and incubated at 37°c until extensive cpe was observed. the cells were harvested using a cellscraper, and suspensions were clarified by centrifugation at 3000×g for table 3 immunogenicity of mtase-defective rmv-hu191 mutants in cotton rats a . inoculum a % infected animals viral titer (log 10 pfu/g) b rmv-hu191 0 nd rmv-hu191-g1788a 0 nd rmv-hu191-g1792a 0 nd dmem 100 4.02 ± 0.37 nd: not detected. a cotton rats were intranasally inoculated with 1.0 × 10 6 pfu of rmv-hu191 or mutants. at 28 days post-infection, rats were challenged with 1 × 10 7 pfu of rmv-hu191. at day 4 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. 20 min at 4°c in an allegra 6 r centrifuge (beckman coulter). the cell pellets were resuspended in 2 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 30,000×g in a beckman ty 50.2 rotor for 2 h, and resuspended in 0.3 ml of dmem, aliquoted, and stored. viral titer was determined by plaque assay. twenty 4-6 week-old female specific-pathogen-free (spf) cotton rats (envigo, indianapolis, in) were randomly divided into four groups (5 cotton rats per group), and housed within the ular facilities at the ohio state university according to iacuc policies and guidelines (animal protocol no. 2009a0221). each inoculated group was separately housed in rodent cages under biosafety level 2 conditions; rats were anesthetized with isoflurane before virus inoculation. cotton rats in groups 1-3 were inoculated with parental rmv-hu191, rmv-hu191-g1788a, and rmv-hu191-g1792a, respectively. cotton rats in group 4 were mock-infected with dmem, and served as uninfected controls. each cotton rat was inoculated intranasally with 5 × 10 6 pfu of virus in a volume of 100 µl. at 4 dpi, cotton rats were sacrificed and lungs were collected for virus titration and rt-pcr. for the immunogenicity study, twenty five 4-6 week-old cotton rats (envigo) were randomly divided into five groups (5 cotton rats per group). cotton rats in groups 1 were mock-infected with dmem and served as uninfected unchallenged control. cotton rats in groups 2, 3 and 4 were intranasally inoculated with 1.0 × 10 6 pfu of rmv-hu191, rmv-hu191-g1788a, and rmv-hu191-g1792a, respectively. cotton rats in groups 5 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 4 weeks post-immunization, the cotton rats in groups 2-5 were challenged with 1.0 × 10 7 pfu of parental rmv-hu191 via intranasal route, and evaluated twice daily for the presence of any clinical symptoms. at 4 days post-challenge, all cotton rats were euthanized by co 2 asphyxiation, and their lungs were collected for virus titration. the immunogenicity of rmv-hu191 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 56°c for 30 min. two-fold dilutions of the serum samples were mixed with an equal volume of dmem containing approximately 100 pfu/well rmv-hu191 in a 96-well plate, and the plate was incubated at room temperature for 1 h with constant rotation. the mixtures were then transferred to confluent vero cells in a 6-well plate in triplicate. after 1 h of incubation at 37°c, the virus-serum mixtures were removed and the cell monolayers were covered with 2.5 ml of eagle's minimal essential media (mem) containing 2% agarose, 0.75% sodium bicarbonate(nahco3), 5% fbs, 20 nm hepes, 2 mm l-glutamine, and 4 mg/ml of streptomycin. then, the cells were incubated for another 6 days before virus plaque titration as described above. the plaques were counted, and 50% 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 8.0). p value of < 0.05 was considered statistically significant. measles and measles vaccination: a review generation of bovine respiratory syncytial virus (brsv) from cdna: brsv ns2 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 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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-2 inhibitors and their combinations in approved drugs to tackle covid-19 pandemic date: 2020-10-20 journal: biorxiv doi: 10.1101/2020.04.23.055756 sha: doc_id: 288644 cord_uid: ywaefpe8 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-2 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-2-induced cytopathic effect and viral replication in vitro. among the potential 72 antivirals tested herein that were previously proposed to inhibit sars-cov-2 infection, only 18% had in vitro antiviral activity. moreover, only eight families had an ic50 below 25 µm or 102 iu/ml. these include chloroquine derivatives and remdesivir, along with plitidepsin, cathepsin inhibitors, nelfinavir mesylate hydrate, interferon 2-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-2 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-2-induced cytopathic effect and viral replication in vitro. among the potential 72 antivirals tested herein that were previously proposed to inhibit sars-cov-2 infection, only 18% had in vitro antiviral activity. moreover, only eight families had an ic50 below 25 µm or 10 2 iu/ml. these include chloroquine derivatives and remdesivir, along with plitidepsin, cathepsin inhibitors, nelfinavir mesylate hydrate, interferon 2-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 2 (sars-cov-2), is causing a respiratory disease pandemic that began in wuhan, china, in november 2019, and has now spread across the world (chen et al., 2020) . to date, remdesivir is the only approved antiviral drug for the specific treatment of this coronavirus infectious disease 2019 or covid-19 (beigel et al., 2020; grein et al., 2020) . however, several drugs are being used in the frontline of clinical management of sars-cov-2-infected individuals in hospitals all around the world, to try to avoid the development of the covid-19 associated pneumonia, which can be fatal. by the end of september 2020, almost a million people had died from covid-19, and over 32 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, 2020) . unfortunately, although remdesivir has proven efficacy in randomized controlled trials (beigel et al., 2020; grein et al., 2020) , 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., 2020) . thus, there is an urgent need to identify novel therapeutic approaches for individuals with covid-19 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-2 replication. compounds were analyzed depending on their expected mechanism of action, to identify candidates tackling diverse steps of the viral life cycle. sars-cov-2 entry requires viral binding and spike protein activation via interaction with the cellular receptor ace2 and the cellular protease tmprss2 (hoffmann et al., 2020) , a mechanism favored by viral internalization via endocytosis. interference with either of these processes has proven to decrease sars-cov-2 infectivity (hoffmann et al., 2020; monteil et al.2020) , and therefore, inhibitors targeting viral entry may prove valuable. in addition, sars-cov-2 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., 2020; mingo et al., 2015; simmons et al., 2005) , providing additional targets for antiviral activity. once sars-cov-2 fuses with cellular membranes, it triggers viral rna release into the cytoplasm, where polyproteins are translated and cleaved by proteases (song et al., 2019) . 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., 2019) . 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., 2019) . 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-1 (hiv-1) and hepatitis c virus (hcv) protease inhibitors, as well as other compounds suggested to have potential activity against sars-cov-2 in molecular docking analysis or in vitro assays. we first assessed the activity of 16 compounds with hypothetical capacity to inhibit viral entry, and then we focused on 22 drugs thought to block viral replication upon sars-cov-2 fusion. molecular docking studies provided an additional 11 candidates, which were predicted to inhibit the sars-cov-2 main protease. finally, 23 compounds with unknown mechanism of action were also assessed. by these means, we have compared 72 drugs and 28 of their combinations for their capacity to counteract sars-cov-2induced cytopathic effect in vitro. we first tested compounds that could have an effect before viral entry by impairing viruscell fusion (supp . table 1) . 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-2 agent wang et al., 2020) . we confirmed the inhibitory effect of hydroxychloroquine against sars-cov-2-induced cellular cytotoxicity on vero e6 cells . a constant concentration of a clinical isolate of sars-cov-2 (id epi_isl_510689) was mixed with increasing concentrations of hydroxychloroquine and added to vero e6 cells. to control for drug-induced cytotoxicity, vero e6 were also cultured with increasing concentrations of hydroxychloroquine in the absence of sars-cov-2. by these means we calculated the concentration at which hydroxychloroquine achieved a 50% maximal inhibitory capacity (ic50). as shown in fig. 1a , this drug was able to inhibit viral-induced cytopathic effects at concentrations where no cytotoxic effects of the drug were observed. the mean ic50 value of this drug in repeated experiments was always below 25 µm (supp . table 1) . these results aligned with previous reports highlighting the in vitro inhibitory capacity of chloroquine derivatives wang et al., 2020) , but contrasted with data on animal models (maisonnasse et al., 2020; rosenke et al., 2020) or currently ongoing clinical trials that have failed to detect any associated benefit of hydroxychloroquine treatment (boulware et al., 2020; cavalcanti et al., 2020) . since hydroxychloroquine was first administered in combination with the antibiotic azithromycin (gautret et al., 2020) , which induces antiviral responses in bronchial epithelial cells (gielen et al., 2010) , we further tested the activity of this compound in our assay. however, in the vero e6 model, azithromycin did not show any antiviral effect (fig. 1a) , and the combination of hydroxychloroquine with azithromycin had a similar activity to that of the chloroquine derivative alone (fig. 1a) . indeed, this was also the case when we tested hydroxychloroquine in combination with different hiv-1 protease inhibitors and other relevant compounds currently being tested in clinical trials (supp. table 2 ). 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-2-induced cytotoxicity assay (supp . table 1) . indeed, interference with clathrinmediated endocytosis is one of the potential mechanisms by which hydroxychloroquine may exert its therapeutic effect against sars-cov-2 . one of these compounds was amantadine, which blocks coated pit invaginations at the plasma membrane (phonphok and rosenthal, 1991) 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., 1993) . when we assessed the antiviral efficacy of these clathrin inhibitors against sars-cov-2, we did not find any prominent effect; only a partial inhibition at 100 µm for amantadine (fig. 1b) . the broad cathepsin b/l inhibitor e64-d also showed partial inhibitory activity (fig. 1b) . e64-d exerts activity against viruses cleaved by cellular cathepsins upon endosomal internalization, as previously described using pseudotyped sars-cov-2 (hoffmann et al., 2020) . while these results could not be confirmed using the specific cathepsin b inhibitor ca-074-me due to drug-associated toxicity (supp . table 1) , it is important to highlight that none of these cathepsin inhibitors is approved for clinical use. these data suggest that sars-cov-2 entry in vero e6 partially relies on clathrin-mediated endocytosis and cellular cathepsins, which cleave the viral spike protein and allow viral fusion once sars-cov-2 is internalized in endosomes. however, hydroxychloroquine antiviral activity was much more potent than that exerted by amantadine or e-64d ( fig. 1a-b) . recently, it has also been suggested that hydroxychloroquine could block sars-cov-2 spike interaction with gm1 gangliosides (fantini et al., 2020) . gm1 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., 2008) . 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-2 induced cytopathic effect (fig. 1b) , as previously reported for sars-cov (lu et al., 2008) . removal of cholesterol redirected ace2 receptor to other domains, but did not alter the expression of the viral receptor (lu et al., 2008) . moreover, nb-dnj, an inhibitor of ganglioside biosynthesis pathway, also decreased sars-cov-2 cytopathic effect (fig. 1b) . these results highlight the possible role of gangliosides in viral binding, although the polar head group of gm3 ganglioside (3' sialyllactose) was not able to reduce viral-induced cytopathic effect (supp. table 1 ). agents involved in autophagy, such as the becn1-stabilizing compounds niclosamide or ciclesonide, inhibit the release of infectious sars-cov-2 to the supernatant (gassen et al., 2020) or reduce the expression of viral nucleoprotein 24 h post-infection (jeon et al., 2020) . however, these autophagy inhibitors were highly toxic in our three-day assay (supp . table 1 ). 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., 2018) . although arbidol has exhibited in vitro efficacy against sars-cov-2 (li, 2020) it showed drug associated cytotoxicity in our assay (supp. table 1 ). we also tested the antiviral activity of two jak inhibitors: baricitinib and tofacitinib. baricitinib was previously suggested to reduce viral entry by interfering with ap2-associated protein kinase 1 (aak1) necessary for clathrin mediated endocytosis stebbing et al., 2020) . however, neither this compound or tofacitinib protected vero e6 cells from sars-cov-2 induced cytopathic effect (supp. table 1 ). 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-2 spike priming on the plasma membrane of human pulmonary cells and avoid viral fusion (hoffmann et al., 2020) . camostat showed no antiviral effect on vero e6 cells (fig. 1c) , 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 1-antitrypsin (att), used to treat severe att human deficiency, was able to exert an antiviral effect on vero e6. however, it required high concentrations that will most likely rely on the activity of these proteases in the endosomal route (fig. 1c) . in order to confirm that previously identified compounds listed in supp. table 1 specifically inhibit the viral entry step, we next employed a luciferase-based assay using pseudotyped lentivirus expressing the spike protein of sars-cov-2, which allows to detect viral fusion on hek-293t cells transfected with ace2. as a control, we used the same lentiviruses pseudotyped with a vsv glycoprotein, where no entry inhibition above 20% was detected for any of the drugs tested (data not shown). in sharp contrast, sars-cov-2 pseudoviruses were effectively blocked by most of the drugs previously tested on vero e6 with wild-type virus (fig. 1d) . the main differences were observed with ca-074-me, ciclesonide and arbidol. these compounds showed a partial blocking effect on ace2 hek-293t cells that was not obvious when using replication competent sars-cov-2 on vero e6 (supp. fig. 1 ). in addition, nb-dnj failed to block viral entry ( fig. 1d ), suggesting that ganglioside dependence may be reduced in ace2 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-2 viral systems, we could identify chloroquine derivatives, cathepsin inhibitors and cholesterol depleting agents as the most promising candidates to block sars-cov-2 endocytosis in vero e6 and hek-293t cells transfected with ace2. however, chloroquine derivatives were the only ones that displayed an ic50 below 25 µm (supp . table 1) , and were also active abrogating pseudoviral entry into hek-293t cells expressing ace2 (fig. 1e) . although camostat failed to inhibit viral fusion on ace2 hek-293t cells (fig. 1e) , its activity was rescued when these cells were transfected with tmprss2. the opposite effect was observed for chloroquine, which reduced its inhibitory activity on tmprss2 transfected cells (fig. 1e ). 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., 2020) ; however, camostat effectively blocked this entry (hoffmann et al., 2020) . our results highlight that alternative routes govern sars-cov-2 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-2 after viral entry and has already been approved for the treatment of covid-19 by the fda and ema. we further confirmed the in vitro capacity of remdesivir to inhibit sars-cov-2induced cytopathic effect on vero e6 ( fig. 2a) . the mean ic50 value of this drug in repeated experiments was always below 10 µm (supp. table 2 ). in combination with hydroxychloroquine, however, remdesivir did not significantly modified its own antiviral effect ( fig. 2b) , 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 2 ). of note, other rna polymerase inhibitors such as galdesivir, which was proposed to tightly bind to sars-cov-2 rna-dependent rna polymerase (elfiky, 2020) , showed no antiviral effect (supp . table 3 ). favipiravir, approved by the national medical products administration of china as the first anti-covid-19 drug in china (tu et al., 2020) , showed only partial inhibitory activity at the non-toxic concentration of 100 µm (supp . table 3) . we also assessed clinically approved protease inhibitors with potent activity against hiv-1. however, none of the hiv-1 protease inhibitors detailed in supp. table 3 showed remarkable protective antiviral activity against sars-cov-2 infection on vero e6 cells, with the exception of nelfinavir mesylate hydrate, which showed an ic50 value below 10 µm (supp . table 3 and fig. 2c ). lopinavir and tipranavir inhibited sars-cov-2induced cytopathic effect at the non-toxic concentration of 20 µm, and amprenavir exhibited activity at the non-toxic concentration of 100 µm (fig. 2c) . darunavir, which is currently being tested in ongoing clinical trials, showed partial inhibitory activity at 100 µm, although this concentration had 8.5 ± 6.2 % of cytotoxicity associated (fig. 2c) . of note, we tested hiv-1 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-2 (supp. fig. 1) . these results indicate that future clinical trials should contemplate the limited antiviral effect displayed by these anti-hiv-1 inhibitors against sars-cov-2 in vitro. we also assessed the inhibitory capacity of hcv protease inhibitors, but none showed any antiviral activity (supp. table 3 ). of note, exogenous interferons 2 alpha and gamma displayed antiviral activity against sars-cov-2 (supp. table 3 ). in light of these results, we tested the inhibitory effect of the tlr 7 agonist vesatolimod that triggers interferon production. although this agonist was not able to protect from the viralinduced cytopathic effect on vero e6 (supp . table 3) , as expected since it is an interferon-producer deficient cell line (emeny and morgan, 1979) , it could still be useful in other competent cellular targets. since severe covid-19 patients display impaired interferon responses (hadjadj et al., 2020) , 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 3cl protease of sars-cov-2, but none of them were effective to protect vero e6 from viral induced cytopathic effect (supp. table 4) . the most potent antiviral tested was plitidepsin (fig. 2d) , which targets the eukaryotic elongation factor 1a2 (eef1a2) and has been previously used for the treatment of multiple myeloma. the mean ic50 value of this drug in repeated experiments was always in nm concentrations (supp . table 3 ). in combination with other active antivirals, we did not observe a reduction on ic50 values (supp. table 2 ). this result indicates no significant synergy, but also highlights the possibility of using plitidepsin without reducing its antiviral activity in combined therapies (fig. 2d) , what could be relevant to avoid possible selection of resistant viruses. overall, plitidepsin showed the lowest ic50 values of all the compounds tested in this in vitro screening ( table 1) . 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-2 infection (supp . table 5 ). such was the case of ivermectin, an fda-approved broad spectrum anti-parasitic agent previously reported to inhibit the replication of sars-cov-2 in vitro as measured by rna accumulation (caly et al., 2020) . 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 28170 and npo compounds. itraconazole, an antifungal that may interfere with internal sars-cov-2 budding within infected cells (wu et al., 2020) , displayed an ic50 value of 80 µm ( fig. 3a and supp. table 5 ). fenofibrate is clinically used to treat dyslipidemia via activation of ppara, and also inhibited the cytopathic effect exerted by sars-cov-2 on vero e6 at 20 µm ( fig. 3b and supp. table 5 ). 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-2 were added to ace-2expressing hek-293t cells in the presence of fenofibrate, viral entry was abrogated (fig. 3c) . the most potent agent found was mdl 28170, a calpain iii inhibitor in a pre-clinical stage of development that displayed activity in the nanomolar range ( fig. 3d and supp. (riva et al., 2020) . moreover, three out of four different calpain and cathepsin inhibitors named npo showed potent antiviral activity too (supp. figure 2) . of note, in combination with other active antivirals, we did not observe a reduction on ic50 values of mdl 28170 (supp. table 2) . inhibitors of calpains, which are cysteine proteases, might impair the activity of viral proteases like 3cl (main protease) and plpro (papain-like protease) (riva et al., 2020; schneider et al., 2012) . however, calpain inhibitors may also inhibit cathepsin bmediated processing of viral spike proteins or glycoproteins, including sars-cov and ebola (schneider et al., 2012; zhou and simmons, 2012) . to understand the mechanisms of action of calpain and cathepsin inhibitors such as mdl 28170, we added lentiviruses pseudotyped with the spike protein of sars-cov-2 to ace-2-expressing hek-293t cells and the same cells also expressing tmprss2 in the presence of this drug. importantly, mdl 28170 only blocked viral entry in ace-2-expressing cells (fig. 3e) . this result indicates that mdl 28170 blocks cathepsins that are implicated in sars-cov-2 entry via the alternative endosomal pathway, as described for chloroquine derivatives and e-64d (fig. 3e) , which are all active when tmprss2 is not present and their inhibitor camostat displays no activity (fig. 3e) . in conclusion, among the 72 compounds and their 28 combinations tested herein for their potential capacity to abrogate sars-cov-2 cytopathic effect, we only found 22 compounds with antiviral activity, and only eight types of these drugs had an ic50 below 25 µm or 10 2 iu/ml ( table 1) . these eight families of compounds were able to abrogate sars-cov-2 release to the supernatant in a dose dependent manner (fig. 4) , 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-2 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 28179 and npo, nelfinavir mesylate hydrate, interferon 2a, 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 28170 or other cathepsin inhibitors to block viral entry via the endosomal pathway in kidney cell lines such as vero e6 or hek-293t. 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-2 entry in pulmonary tissues or in cells expressing tmprss2. these findings can explain why randomized clinical trials using hydroxychloroquine have failed to show a significant protective effect (boulware et al., 2020; cavalcanti et al., 2020) . nonetheless, in combined therapies, it should be noted that agents targeting the alternative endosomal sars-cov-2 entry route such as hydroxychloroquine or mdl 28170 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 2500 patients, hydroxychloroquine treatment showed a significant reduction of in-hospital mortality (arshad et al., 2020) . thus, since alternative routes govern sars-cov-2 viral entry depending on the cellular target (ou et al., 2020a) , effective treatments might be needed to block both plasma membrane fusion and endosomal entry to broadly achieve viral suppression. sars-cov-2 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 ic50 values of drugs obtained in vitro may not reflect what could happen in vivo upon sars-cov-2 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-2 , while also questioning the use of hydroxychloroquine in monotherapy (maisonnasse et al., 2020) . 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-2-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 5% fetal calf serum (fcs; euroclone), 100 u/ml penicillin, 100 µg/ml streptomycin, and 2 mm glutamine (all thermofisher scientific). hek-293t (atcc repository) were maintained in dmem with 10% fetal bovine serum, 100 iu/ml penicillin and 100 µg/ml streptomycin (all from invitrogen). hek-293t overexpressing the human ace2 were kindly provided by integral molecular company and maintained in dmem (invitrogen) with 10% fetal bovine serum, 100 iu/ml penicillin and 100 µg/ml streptomycin, and 1 µg/ml of puromycin (all from invitrogen). tmprss2 human plasmid (origene) was transfected using x-tremegene hp transfection reagent (merck) on hek-293t overexpressing the human ace2 and maintained in the previously described media containing 1 mg/ml of geneticin (invitrogen) to obtain tmprss2/ace2 hek-293t cells. virus isolation, titration and sequencing. sars-cov-2 was isolated from a nasopharyngeal swab collected from an 89-year-old male patient giving informed consent and treated with betaferon and hydroxychloroquine for 2 days before sample collection. the swab was collected in 3 ml medium (deltaswab vicum) to reduce viscosity and stored at -80ºc until use. vero e6 cells were cultured on a cell culture flask (25 cm 2 ) at 1.5 x 10 6 cells overnight prior to inoculation with 1 ml of the processed sample, for 1 h at 37ºc and 5% co2. afterwards, 4 ml of 2% fcs-supplemented dmem were supplied and cells were incubated for 48 h. supernatant was harvested, centrifuged at 200 x g for 10 min to remove cell debris and stored at -80ºc. cells were assessed daily for cytopathic effect and the supernatant was subjected to viral rna extraction and specific rt-qpcr using the sars-cov-2 upe, rdrp and n assays (corman et al., 2020) . the virus was propagated for two passages and a virus stock was prepared collecting the supernatant from vero e6. 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-2 panel (swift biosciences). sequencing ready libraries where then loaded onto illumina miseq platform and a 300bp 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, 2011) . sequencing reads were then mapped against coronavirus reference (nc_045512.2) using bowtie2 tool (langmead, b. and salzberg, s, 2012) . consensus genomic sequence was called from the resulting alignment at a 18x1800x879 average coverage using samtools (li et al., 2009) . genomic sequence was deposited at gisaid repository (http://gisaid.org) with accession id epi_isl_510689. pseudovirus production. hiv-1 reporter pseudoviruses expressing sars-cov-2 spike protein and luciferase were generated using two plasmids. pnl4-3.luc.r-.e-was obtained from the nih aids repository. sars-cov-2.sctδ19 was generated (geneart) from the full protein sequence of sars-cov-2 spike with a deletion of the last 19 amino acids in c-terminal, human-codon optimized and inserted into pcdna3.4-topo (ou et al., 2020b) . spike plasmid was transfected with x-tremegene hp transfection reagent µm to 0.0512 nm at ⅕ serial dilutions. plitidepsin was also assayed at concentrations ranging from 10 µm to 0.5 nm at 1/3 dilutions. interferons were assayed at concentrations ranging from 10 4 to 0.0005 iu/ml at ⅕ serial dilutions. when two drugs were combined, each one was added at a 1:1 molar ratio at concentrations ranging from 100 µm to 0.0512 nm at ⅕ serial dilutions. in combination with other drugs, plitidepsin was also assayed at concentrations ranging from 10 µm to 0.5 nm at 1/3 dilutions. e6 cells together with 10 1.8 tcid50/ml of sars-cov-2, a concentration that achieves a 50% 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 e6 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 3 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 100% of viability to normalize data and calculate the percentage of cytopathic effect. statistical differences from 100% were assessed with a one sample t test. all analyses and figures were generated with the graphpad prism v8.0b software. in silico drug modeling. we performed glide docking using an in-house library of all approved drug molecules on the 3cl protease of sars-cov-2. for this, two different receptors were used, the 6lu7 pdb structure, after removing the covalently bound inhibitor, and a combination of two crystals from the diamond collection (https://www.diamond.ac.uk/covid-19). receptors were prepared with the schrodinger's protein wizard and glide sp docking was performed with two different hydrogen bond constraints: glu16 and his163 (with epsilon protonation); we enforced single constraints and also attempted the combination of both. the best 20 molecules, based on glides's docking score were selected. top docking scores, however, did not exceed -9, indicating poor potential binding. pseudovirus assay. hek-293t overexpressing the human ace2 and tmprss2 were used to test antivirals at the concentrations found to be effective for sars-cov-2 without toxicity, which were the following: 5 µm for niclosamide; 10 µm for chloroquine, chlorpromazine, ciclesonide, mdl 28170 and fenofibrate; 20 µm for hydroxychloroquine, ca-074-me and arbidol hcl; 25 µm for e-64d; 50 µm for baricitinib; 100 µm for amantadine, nb-dnj, 3' sialyl-lactose na salt, tofacitinib, and camostat mesylate; 1000 µm for methyl-b-cyclodextrin, and 12,5 mg/ml for att. a constant pseudoviral titer was used to pulse cells in the presence of the drugs. 48h postinoculation, cells were lysed with the glo luciferase system (promega). luminescence was measured with an ensight multimode plate reader (perkin elmer). table 1 . compounds with antiviral activity grouped in colors depending on their ic50 values, expressed in µm unless otherwise indicated. activity of hydroxychloroquine and azithromycin. cytopathic effect on vero e6 cells exposed to a fixed concentration of sars-cov-2 in the presence of increasing concentrations of hydroxychloroquine, azithromycin, and their combination. drugs were used at a concentration ranging from 0.0512 nm to 100 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 ic50 value of this graph is indicated. cytotoxic effect on vero e6 cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). b. cytopathic effect on vero e6 cells exposed to a fixed concentration of sars-cov-2 in the presence of increasing concentrations of amantadine, a clathrin-mediated endocytosis inhibitor, e-64d, 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 0.0512 nm to 100 µm aside from methyl-b-cyclodextrin, which was used 10 times more concentrated. nonlinear fit to a variable response curve from one experiment with two replicates is shown (red lines). cytotoxic effect on vero e6 cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). c. cytopathic effect on vero e6 cells exposed to a fixed concentration of sars-cov-2 in the presence of increasing concentrations of camostat, a tmprss2 inhibitor, and att, an alfa-1 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-2 spike in ace2 expressing hek-293t cells. values are normalized to luciferase expression by mock-treated cells set at 100%. mean and s.e.m. from two experiments with two replicates. cells were exposed to fixed amounts of sars-cov-2 spike lentiviruses in the presence of a non-toxic constant concentration of the drugs tested on vero e6. statistical deviations from 100% were assessed with a one sample t test. e. comparison of entry inhibitors blocking viral endocytosis, such as chloroquine, with inhibitors blocking serine protease tmprss2 expressed on the cellular membrane, such as camostat, on different cell lines. ace2 expressing hek-293t cells transfected or not with tmprss2 were exposed to sars-cov-2 spike lentiviruses as described in b. values are normalized to luciferase expression by mock-treated cells set at 100%. mean and s.e.m. from at least two representative experiments with two replicates. statistical deviations from 100% were assessed with a one sample t test. a. cytopathic effect on vero e6 cells exposed to a fixed concentration of sars-cov-2 in the presence of increasing concentrations of remdesivir. drug was used at a concentration ranging from 0.0512 nm to 100 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 ic50 value of this graph is indicated. cytotoxic effect on vero e6 cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). b. cytopathic effect on vero e6 cells exposed to a fixed concentration of sars-cov-2 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 0.0512 nm to 100 µm (left panel). alternatively, remdesivir was used at a concentration ranging from 0.0512 nm to 100 µm at the fixed indicated concentrations of hydroxychloroquine (right panel). c. cytopathic effect on vero e6 cells exposed to a fixed concentration of sars-cov-2 in the presence of increasing concentrations of protease inhibitors against hiv-1. nelfinavir mesylate hydrate was the only drug with activity. inhibitors were used at a concentration ranging from 0.0512 nm to 100 µm. the particular ic50 value of this graph is indicated d. cytopathic effect on vero e6 cells exposed to a fixed concentration of sars-cov-2 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 0.5 nm to 10 µm. the particular ic50 value of these graphs is indicated. cytopathic effect on vero e6 cells exposed to a fixed concentration of sars-cov-2 in the presence of increasing concentrations of itraconazole. drug was used at a concentration ranging from 0.0512 nm to 100 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 ic50 value of this graph is indicated. cytotoxic effect on vero e6 cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). b. cytopathic effect on vero e6 cells exposed to a fixed concentration of sars-cov-2 in the presence were assessed with a one sample t test. supp. table 1 . antiviral activity of potential entry inhibitors tested against sars-cov-2. na; not active. ic50 values are reported in µm unless otherwise indicated. supp. table 2 . antiviral activity of potential inhibitors against sars-cov-2 tested in combination. na; not active. table 3 . antiviral activity of potential post-entry inhibitors against sars-cov-2. na; not active. ic50 values are reported in µm unless otherwise indicated. supp. table 4 . antiviral activity of potential inhibitors against sars-cov-2 with predicted capacity to block sars-cov-2 viral protease. na; not active. supp. table 5 . antiviral activity of potential inhibitors against sars-cov-2 with unknown mechanism of action. na; not active. cytopathic effect on vero e6 cells exposed to a fixed concentration of sars-cov-2 in the presence of increasing concentrations of hiv-1 reverse transcriptase inhibitors. drugs were used at a concentration ranging from 0.0512 nm to 100 µm. non-linear fit to a variable response curve from one experiment with two replicates is shown (red lines). cytotoxic effect on vero e6 cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). cytopathic effect on vero e6 cells exposed to a fixed concentration of sars-cov-2 in the presence of increasing concentrations of calpain and cathepsin inhibitors npo. drugs were used at a concentration ranging from 0.0512 nm to 100 µm. non-linear fit to a variable response curve from one experiment with two replicates is shown (red lines). cytotoxic effect on vero e6 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 (ep20382821.5). the authors declare that no other competing financial interests exist. treatment with hydroxychloroquine, azithromycin, and combination in patients hospitalized with covid-19 remdesivir for the treatment of covid-19 -preliminary report a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid-19 the fdaapproved drug ivermectin inhibits the replication of sars-cov-2 in vitro hydroxychloroquine with or without azithromycin in mild-to-moderate covid-19 epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir against sars-cov-2 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-2 infection analysis of sars-cov-2-controlled autophagy reveals spermidine hydroxychloroquine and azithromycin as a treatment of covid-19: 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-19 impaired type i interferon activity and inflammatory responses in severe covid-19 patients histopathological findings and viral tropism in uk patients with severe fatal covid-19: a post-mortem study arbidol (umifenovir): a broad-spectrum antiviral drug that inhibits medically important arthropod-borne flaviviruses the novel coronavirus 2019 (2019-ncov) uses the sarscoronavirus receptor ace2 and the cellular protease tmprss2 for entry into target cells insights from nanomedicine into chloroquine efficacy against covid-19 identification of antiviral drug candidates against sars-cov-2 from fda-approved drugs (microbiology) fast gapped-read alignment with bowtie 2 an exploratory randomized controlled study on the efficacy and safety of lopinavir/ritonavir or arbidol treating adult patients hospitalized with mild/moderate covid-19 (elacoi) the sequence alignment/map format and samtools hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro lipid rafts are involved in sars-cov entry into vero e6 cells hydroxychloroquine use against sars-cov-2 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 npc1 + endolysosomes is a rate-defining step inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2. 29 hydroxychloroquine-mediated inhibition of sars-cov-2 entry is attenuated by tmprss2 (microbiology) characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov repurposed antiviral drugs for covid-19 -interim who solidarity trial results stabilization of clathrin coated vesicles by amantadine, tromantadine and other hydrophobic amines baricitinib as potential treatment for 2019-ncov acute respiratory disease discovery of sars-cov-2 antiviral drugs through large-scale compound repurposing hydroxychloroquine proves ineffective in hamsters and macaques infected with sars-cov-2 (pharmacology and toxicology) severe acute respiratory syndrome coronavirus replication is severely impaired by mg132 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-19: combining antiviral and anti-inflammatory treatments a review of sars-cov-2 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 (2019-ncov) in vitro solidarity" clinical trial for covid-19 treatments clinical benefit of remdesivir in rhesus macaques infected with sars-cov-2 (microbiology) analysis of therapeutic targets for sars-cov-2 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-283309-ovx5fzsg 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-hku2-like swine enteric alphacoronavirus (seacov) infection in cultured cells and development of a seacov infectious clone date: 2019-08-09 journal: virology doi: 10.1016/j.virol.2019.08.006 sha: doc_id: 283309 cord_uid: ovx5fzsg swine enteric alphacoronavirus (seacov), also known as swine acute diarrhea syndrome coronavirus (sads-cov), belongs to the species rhinolophus bat coronavirus hku2. herein, we report on the primary characterization of seacov in vitro. four antibodies against the seacov spike, membrane, nucleocapsid and nonstructural protein 3 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-10 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 ns7a and ns7b 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., 2017; pan et al., 2017; zhou et al., 2018) . this emerging virus was first isolated from clinically sick animals in commercial swine herds at guangdong province, china during february-may 2017. the mortality rate in less than 5 days old piglets was over 90%, whereas it dropped to 5% in piglets older than 8 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 hku2 identified in the same region a decade earlier (lau et al., 2007) . a retrospective study indicated that the virus had emerged in guangdong since august 2016 . the isolated virus was infectious to pigs and cause mild or severe diarrhea symptom when inoculated orally into conventional newborn piglets xu et al., 2019; zhou et al., 2018) . 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 27.2 kb in size with the gene order of 5'-orf1a/1b (orf1ab)-spike (s)-orf3-envelope (e)-membrane (m)-nucleocapsid (n)-ns7a/ns7b-3'. seacov shared 95% nucleotide (nt) sequence identity with the bat cov hku2 strains and 96-98% nt identity with the hku2-derived bat sadsrelated coronavirus (sadsr-cov) strains at the complete genome level zhou et al., 2018) . interestingly, seacov and other hku2-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., 2019; wang et al., 2015 wang et al., , 2017b between α-cov and β-cov (lau et al., 2007; pan et al., 2017) . the cov genome harbors a few genus-specific accessory genes within the 3'-part genomic region encoding the four structural proteins (s-e-m-n) . it is found that seacov contains a putative open reading frame (orf), ns7a, and a downstream ns7b orf (overlapped with ns7a) after the n gene at the 3'-end genome (lau et al., 2007; pan et al., 2017) . the ns7a is shared by the hku2 and seacov strains, whereas ns7b 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., 2014) . for examples, the severe acute respiratory syndrome coronavirus (sars-cov) orf-3a was found to induce necrotic cell death, lysosomal damage and caspase-1 activation, which largely contribute to the clinical manifestations of sars-cov infection (yue et al., 2018) . in addition, sars-cov orf6 and orf7b may also be also associated with the virulence. in another newly emerged swine cov, pdcov, its accessory ns6 protein has been reported to counteract host innate antiviral immune response by inhibiting ifn-β production that interacts with rig-i/mda5 (fang et al., 2018) . whether the predicted ns7a and ns7b 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. 2.1. 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 3 (nsp3) acidic domain (ac) of orf1a, were expressed as soluble products in the bacteria; the seacov spike subunit 1 (s1) was expressed in insect cells, secreting into the cultured medium. purified recombinant seacov proteins (n, s1 and ac) and an antigenic peptide corresponding to the last 14 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 48 h post-infection (hpi) using respective pab showed that the four viral antigens (n, m, s1 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. 1a) . in contrast, mock-infected controls did not show any positive ifa signals (fig. 1a) . 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 4, 8, 12, and 24 hpi, and labeled with four pab, respectively. perinuclear and cytoplasmic foci were detected by anti-n staining at 4 and 8 hpi, and were distributed throughout the cytoplasm at 12 and 24 hpi, probably reflecting that n protein is associated with sites of viral rna replication in early infection phase and assembled into virions subsequently (fig. 1b) . anti-ac (nsp3) staining also resulted in detection of perinuclear foci at four time points, indicating localization to the viral replication-transcription complexes (fig. 1c) , which was similar to the pattern of nsp3 antibody observed in sars-cov-infected vero cells (prentice et al., 2004) . confocal microscopy detected discrete cytoplasmic fluorescence signal throughout the cytoplasm with anti-m (fig. 1d ) and anti-s1 (fig. 1e ) as early as 4 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 42 kda in the lysate of seacov-infected cells but not in control cells at 48 hpi by western blot analysis (fig. 1f ). 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. 1f) . expression of the m protein with the predicted 25-kda molecular size was also detected by using anti-m pab in seacov-infected cells (fig. 1g ). the reactivity of anti-s1 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., 2014) . 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 (~16 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 27.2-kb seacov genomic cdna from the passage-10 virus ("seacov-p10") by a single step ligation of 15 overlapping fragments into a bac expression vector, resulting in a full-length cdna clone of seacov named psea ( fig. 2a) . 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 (a24222t and g24223c) in orf3 were introduced in psea as a genetic marker to distinguish the parental virus seacov-p10 ( fig. 2a) . bhk-21 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-21 cells were inoculated onto fresh vero cells at 2-3 days post-transfection. seacov-induced cytopathic effects (cpe) were visualized at 48 hpi in inoculated vero cells; viral antigens were detected by ifa using anti-n, anti-m, anti-s1 or anti-ac to stain cells, confirming the successful recovery of recombinant seacov (rseacov; fig. 2b ). 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. 2c ). 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 100-120 nm in diameter with surface projections (fig. 2d) , consistent with our previous report of seacov isolation in vero cells . the comparative growth kinetics of rseacov and the parental seacov-p10 were analyzed by infection of vero cells with the respective virus at the same multiplicity of infection (moi) of 0.1. the infectious virus titers were determined at different time points postinfection (2, 6, 12, 24, 36, 48, 60 and 72 hpi) . the result showed that rseacov had the growth kinetics similar to the parental seacov-p10 ( fig. 2e ). of note, the maximal rates of seacov-p10 or rseacov production were from 6 to 12 hpi, suggesting that the exponential release of virus occurred before 6 hpi, which was consistent with detection of n, m, s and ac expression as early as 4 hpi ( fig. 1b-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 4-6 h (prentice et al., 2004; qin et al., 2019) . 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 orf3 [in tgev , sars-cov (yount et al., 2005) , pedv (ji et al., 2018) or human cov nl63 (donaldson et al., 2008) ] and the gene 7 [in tgev (ortego et al., 2003) ] are dispensable for propagation in vitro. the corresponding genes, orf3 and ns7a, are also present in the seacov genome; therefore, we will aim to generate reporter virus expressing luciferase or green fluorescent protein by replacement of orf3 or ns7a with the reporter gene in future studies. coronaviruses can produce multiple sgrnas are produced by discontinuous transcription. each sgrna contains a short 5' leader sequence derived from the 5'-end of the genome and a body sequence from the 3'-poly (a) stretching to a position in the upstream of each orf encoding a structural or accessory protein (sola et al., 2015) . the fusion site of the leader and body sequence in each sgrna is termed transcription regulatory sequence (trs). the seacov leader sequence of 75 nt from the 5'-end to the leader trs was proposed according to the previous report (lau et al., 2007) ; 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., 2013) (fig. 3a) . the existence of all predicted subgenomic mrnas (sgrna; mrna 2 to mrna 7) for the expression of s, orf3, e, m, n and ns7a was investigated further (fig. 3b) . 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 (s1-r, sgorf3-r, sge-r, sgm-r, sgn-r and ns7a-r) amplified at least one major band of the expected size by agarose gel electrophoresis analysis (fig. 3c ). the appearance of multiple pcr bands was in line with what was expected, since except for the primers lf and s1-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-s1-pab and an anti-ac-pab, respectively (magnification = 200×). alexa fluor 488-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 s1 detection using an olympus confocal microscope. vero cells infected with seacov were fixed at 4, 8, 12, and 24 hpi, and labeled with four pabs, respectively. bar = 10 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 6, could also amplify those of mrnas 2 to 5, resulting in detection of five bands (fig. 3c) . 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. 3d) . we also noticed that both orfs of ns7a and ns7b are connected with a body trs in the upstream, implying a bicistronic mrna encoding ns7a and ns7b (fig. 3e ). since amplification with the reverse primer ns7a-r could not cover the entire ns7b, we next determined whether a potential ns7b sgrna is present using the leader primer lf and a new reverse primer ns7-r corresponding to the 3'-end of orf7b by rt-pcr. a single band of approximately 400-bp was amplified by optimizing the pcr condition and detected by agarose gel electrophoresis analysis; the other smaller bands were not found (fig. 3e) . sequence analysis revealed that the trs for this bicistronic sgrna ns7 was exactly aacuaaa and one nt upstream of the aug start codon of ns7a, which is consistent with the prediction (fig. 3e) . we further expressed and purified the complete ns7a or ns7b gene in the bacteria. both products were found in the inclusion bodies. however, the resulting anti-ns7a or anti-ns7b 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 ns7a and ns7b are either, not highly antigenic or the denatured antigens used to generate pabs destroy the native protein structure. development of monoclonal antibodies against ns7a and ns7b 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-p10 in vero cells. cells were infected in triplicate with virus at a moi = 0.1. cells were harvested at 2, 6, 12, 24, 36, 48, 60 and 72 hpi, and virus titers (tcid 50 /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., 2004; gosert et al., 2002; qin et al., 2019; salanueva et al., 1999; v'kovski et al., 2015) . dmvs are membrane structures where viral genomic rna is recognized by the host cell machinery and translated into non-structural proteins (orf1ab), assembling into viral replication-transcription complexes (gosert et al., 2002) , 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 (s1-r, sgorf3-r, sge-r, sgm-r, sgn-r and ns7a-r/ns7-r) used for pcr amplification of distinct subgenomic mrnas (sgrnas) are indicated by arrows under the genome. the seven small black boxes at the 5' ends of the genomic rna (grna) and sgrnas depict the common leader sequence. genomic and subgenomic rna numbers (1 for grna and 2 to 7 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. 4a ; 24 hpi) but not in uninfected cells (fig. 4c) . of note, time course analysis of nsp3 detection in fig. 1c 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 0.1) 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 24 hpi and thereafter (fig. 4b) . 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 4-6 h. by rt-pcr analysis, we experimentally identified all proposed seacov sgrnas containing the leader-body junction sites. among six sgrnas, a bicistronic mrna 7 was utilized by the accessory ns7a and ns7b 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-81) and a baby hamster kidney fibroblast cell line, bhk-21 (atcc ccl-10) were grown in dmem supplemented with 10% fetal bovine serum (fbs) and 1% antibiotics at 37°c, respectively. the seacov isolate ch/gd-01/2017 at the passage 10 (p10) used in this study was cultured in vero cells. the virus titers were determined by endpoint dilutions as 50% tissue culture infective dose (tcid 50 ) on vero cells. the control virus pedv (zju/g2/2013 strain; genbank accession no. ku558701) was also cultured in vero cells as described earlier (ji et al., 2018; qin et al., 2017) . vero cells infected by the seacov or pedv (at 24 h postinoculation, hpi) were fixed with 2.5% glutaraldehyde in phosphate buffer (0.1 m, ph 7.0) and 1% oso4 in phosphate. ultrathin sections were prepared as described previously , stained by uranyl acetate and alkaline lead citrate for 5-10 min, and observed using a hitachi model h-7650 tem. polyclonal antibodies (pab) against the spike subunit 1 (anti-s1), membrane (anti-m), nucleocapsid (anti-n) and the nonstructural protein 3 (nsp3) 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 55 days postimmunization (dpi). anti-s1, anti-n and anti-ac pabs of seacov were prepared in-house. briefly, full-length n (1128 nt, 379 aa,~42 kda) or ac (435 nt, 145 aa,~16 kda) of seacov were expressed with a sixhistidine tag in escherichia coli according to methods described previously (huang et al., 2011) , whereas seacov-s1 (1638 nt, 174 aa, 62 kda) with a six-histidine tag was expressed by baculovirus system in sf9 insect cells as described previously (wang et al., 2017a) . the purified proteins were used to immunize rabbits, and antisera were harvested at 55 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 (5'-atagagtccttatcttttt-3') and six gene specific reverse primers, s1-r (5'-caatggcatttctgtg tacctctc-3'), sgorf3-r (5'-agtaatctgcttacaacagc-3'), sge-r (5'-agacattaattatggggcat-3'), sgm-r (5'-gttcgcgttctgcga taaag-3'), sgn-r (5'-atctgcgtgaggaccagtac-3'), ns7a-r (5'-aatctgcaaaatctgccaac-3'), were designed for amplification of all seacov subgenomic mrnas (fig. 3a ) from the obtained cdna with a taq dna polymerase (transgen, beijing, china) in a total volume of 50 μl by pcr. the pcr condition was set at 35 cycles of 94°c for 30 s, 50°c for 30 s, 72°c for 3 min with an initial denaturing of the template dna at 94°c for 3 min and a final extension at 72°c for 5 min. the resulting pcr fragments were analyzed on a 1% agarose gel (fig. 3b) and then subcloned into a peasy-t1 vector (transgen, beijing, china) followed by sanger sequencing. for amplification of the subgenomic mrna 7 containing the entire ns7a/ns7b, the reverse primer ns7-r (5'-ttacgtgcttaccattgtgt-3') was used, and the pcr extension time was shortened to 45 s. analysis of dna sequences was performed using the lasergene package (dnastar inc., madison, wi). the expression vector, designated as psb2μ, used to construct a fulllength seacov cdna clone, was based on a bac backbone vector psmart-bac-bamhi (copyright v2.0 bac cloning kits, lucigen). this psmart-bac vector was modified to insert a yeast replication origin (2μ) from the plasmid pyes2 (invitrogen), a cytomegalovirus (cmv) promoter from the plasmid pcdna3 (invitrogen), a hepatitis delta virus ribozyme (hdvrz) sequence from a prrsv (porcine reproductive and respiratory syndrome virus) infectious clone ptri-53rz-pgxg (huang et al., 2009) , and a bovine growth hormone (bgh) polyadenylation and terminator from the plasmid pcdna3 (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-p10 (27,155 nt) was determined as described previously . briefly, a total of 15 overlapping fragments covering the entire genome was amplified by rt-pcr using the q5 high-fidelity 2×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 2-nt genetic marker on the orf3 gene of the infectious clone, two point mutations, a to t, and g to c at nucleotide positions 24222-24223, corresponding to the seacov-p10 genome, were generated on the fragment s-2 by fusion pcr (fig. 2a) . subsequently, all 14 fragments identical to the consensus sequence together with the mutated s-2 fragment were re-amplified from each clone with primers listed in table 1 . it was then assembled into the expression vector (psb2μ) 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. 2) . 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 prk5 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 dh10b strain using qiaprep miniprep kit (qiagen) and quantified by a nanodrop spectrophotometry. bhk-21 cells were seeded at 2×10 5 per well of a sixwell plate and grown until 60-70% confluence before transfection. one microgram each of psea and prk-n-flag were co-transfected into the cells using lipofectamine 3000 (invitrogen) according to the manufacturer's protocol. transfected cells were cultured for 2-3 days. the supernatant was collected and passaged onto fresh vero cells on 12-well plates and cultured for 3 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 50 . viral particles in the supernatants from rseacov-infected cell cultures were negatively stained and examined under tem. a 1.5-kb dna fragment harboring the introduced mutations in the orf3 gene was amplified by rt-pcr using primers tf21 (5'-tactggatgttgtggcatgt-3') and tr21 (5'-ttccacttaaaatcgtcaga-3'). 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 4% paraformaldehyde in pbs for 20 min and then permeabilized with 0.5% triton x-100 for 10 min. anti-n, anti-m, anti-s1 or anti-ac pab, each at a 1:1000 dilution in pbs, was added over the cells and incubated for 1 h at 37°c. cells were then washed thrice with pbs and alexa fluor 488-labeled goat anti-rabbit igg (thermo fisher scientific) at a 1:1000 dilution was then added. after 30 min of incubation at 37°c, the cells were again washed thrice with pbs followed by 4',6-diamidino-2-phenylindole (dapi) staining, and were visualized under a fluorescence microscope (dmi3000b, leica, germany). for time course analysis of detection of n, m, s1 or ac, fluorescent images were obtained with a confocal laser scanning microscope (fluoviewver fv1000-ix81; olympus, japan). for western blot analysis, seacov-infected cells were lysed in lysis buffer (25 mm tris-hcl, 200 mm nacl, 10 mm naf, 1 mm na 3 vo 4 , 25 mm β-glycerophosphate, 1% np40, 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 3% bovine serum albumin (bsa) overnight at 4°c. proteins were detected using the anti-n pab or anti-m pab at 1:1000 dilution followed by incubation with horseradish peroxidase (hrp)-conjugated anti-rabbit igg (1:5000 dilution; thermo fisher scientific). the consensus sequence of seacov-p10 used for construction of the infectious clone has been deposited in genbank under accession no. mk977618. natural science foundation of china (31872488), and the fundamental research funds for the central universities of china (2019fza6014). 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 nl63 porcine deltacoronavirus accessory protein ns6 antagonizes interferon beta production by interfering with the binding of rig-i/mda5 to double-stranded rna a new bat-hku2-like coronavirus in swine rna replication of mouse hepatitis virus takes place at double-membrane vesicles origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states identification and characterization of a porcine monocytic cell line supporting porcine reproductive and respiratory syndrome virus (prrsv) replication and progeny virion production by using an improved dnalaunched prrsv reverse genetics system expression of the putative orf1 capsid protein of torque teno sus virus 2 (ttsuv2) and development of western 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novel reassortant mammalian orthoreovirus 3 (mrv3) from a diarrheic piglet and seroepidemiological survey of mrv3 in diarrheic pigs from east china structural maturation of the transmissible gastroenteritis coronavirus continuous and discontinuous rna synthesis in coronaviruses engineering the transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity shared common ancestry of rodent alphacoronaviruses sampled globally qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus new insights on the role of paired membrane structures in coronavirus replication the coronavirus nucleocapsid protein is dynamically associated with the replication-transcription complexes development and application of real-time rt-pcr and s1 protein-based indirect elisa for porcine deltacoronavirus emerging and re-emerging coronaviruses in pigs discovery, diversity and evolution of novel coronaviruses sampled from rodents in china discovery of a highly divergent coronavirus in the asian house shrew from china illuminates the origin of the alphacoronaviruses enhancement of safety and immunogenicity of the chinese hu191 measles virus vaccine by alteration of the s-adenosylmethionine (sam) binding site in the large polymerase protein isolation and characterization of a highly pathogenic strain of porcine enteric alphacoronavirus 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-3a 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 (2016yfd0500102), the national key: cord-267613-hsc2x36j 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-2 date: 2020-06-19 journal: biorxiv doi: 10.1101/2020.06.19.161042 sha: doc_id: 267613 cord_uid: hsc2x36j there are an urgent need for antivirals to treat the newly emerged sars-cov-2. to identify new candidates we screened a repurposing library of ~3,000 drugs. screening in vero cells found few antivirals, while screening in human huh7.5 cells validated 23 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-2 in these cells. entry in lung epithelial calu-3 cells is ph-independent and requires tmprss2, while entry in vero and huh7.5 cells requires low ph and triggering by acid-dependent endosomal proteases. moreover, we found 9 drugs are antiviral in lung cells, 7 of which have been tested in humans, and 3 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 (1). first, severe acute respiratory syndrome (sars-cov) emerged in china in 2003, followed by middle east respiratory syndrome (mers-cov) in 2012 (2, 3) . while sars was, in the end eradicated, mers continues to cause infections in the middle east. beginning in december 2019, into continuing into january 2020, 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-2 (4). unfortunately, this virus is highly infectious and has spread rapidly around the world. identification of broadly acting sars-cov-2 antivirals is essential to clinically address sars-cov-2 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-2 in vitro, with promising results in clinical trials (5) (6) (7) . 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-2 (8, 9) . this led to early adoption of these agents to treat covid-19 (the disease caused by sars-cov-2 infection); however, little efficacy of these agents has been demonstrated in subsequent clinical trials (10) . it remains unclear why these agents have not been more active in humans. there are currently more than 3000 fda approved drugs, and many others that have been tested in humans. we created an in-house library of 3059 drugs including ~1000 fda approved drugs and ~2100 drug-like molecules against defined molecular targets with validated pharmacological activity. in addition, we purchased drugs with reported anti-sars-cov-2 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-2 (11). 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-2. they are robustly infected, and thus vero cells are widely used as a model system to screen for antivirals (8, (12) (13) (14) . 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 huh7.5 was readily infected with sars-cov-2. screening in this human cell line we validated 23 drugs that were active in dose-response experiments and showed a favorable selective index versus toxicity (15) . 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-2 has only begun to be elucidated with much of what we know being inferred from studies of the related sars-cov-1 (16, 17) . the coronavirus glycoprotein, or spike, requires proteolytic processing for entry (16, 18, 19) . this processing can occur outside the cell, or within the endolysosomal compartment (16, 19) . both sars-cov-1 and -2 engage angiotensin-converting enzyme 2 (ace2) as their plasma membrane receptor (20) (21) (22) . 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 (23) (24) (25) . since cathepsins require a low ph for activity, chloroquine and its derivatives that neutralize this low ph can effectively block viral entry (23) (24) (25) . recent studies have also identified a plasma membrane-associated serine protease, tmprss2, is active against spike, cleaving the protein extracellularly thereby bypassing the requirement for endosomal proteases (26) (27) (28) . whether sars-cov-2 enters through different routes in different cell types remains unclear. lung epithelial cells are the major cellular target for sars-cov-2 in vivo and have been used to explore the role of tmprss2 in infection. perhaps surprisingly, while we found remdesivir was antiviral in calu-3, hydroxychloroquine was not. since a panel of quinolines had no activity in calu-3 cells, these data suggest that entry in lung epithelial cells is independent of low-ph processing in the endosomal compartment. in contrast, the tmprss2 inhibitor camostat was highly active in calu-3 cells but inactive in vero and huh7.5 cells. these data demonstrate distinct modes of entry in lung cells (21) . further, these data suggest that there may be other fundamentally different cellular requirements in different cell types. we screened our 23 validated candidates in calu-3 cells and found only 9 drugs showed activity, including 3 fda approved drugs: cyclosporine, dacomitinib and salinomycin. in additional studies, we found that cyclosporine analogs that target cyclophilin a were active against sars-cov-2, but not compounds that target calcineurin. identifying broadly acting antivirals is essential to move forward with clinical treatments for sars-cov-2. vero cells are permissive to infection and can be used for antiviral screening for direct acting antivirals sars-cov-2 is routinely propagated in vero e6 cells (12, 14, 26) . when growing the virus in either vero e6 or vero ccl81 cells, two different strains of vero cells from atcc, we observed that sars-cov-2 is cytopathic in vero e6, but not in vero ccl81 (data not shown) (12) . moreover, viral stocks propagated from either of these cells produced similar titers of virus (1x10 7 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-2 more directly in infected cells, and we chose vero ccl81 to uncouple toxicity from infection. we first validated that our antibodies could detect infection of sars-cov-2. we used an antibody to dsrna, and to sars-cov-2 spike (figure 1a ) (29) (30) . we created an in-house library of 3059 drugs purchased from selleckchem. this library contains ~1000 fda approved drugs and ~2100 drug-like molecules against defined molecular targets with validated pharmacological activity. the library contains 678 known kinase inhibitors, 435 annotated cancer therapeutics, 190 epigenetic regulators, 411 anti-viral/infectives, 596 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 1b) (8) . next, we validated the assay metrics, and observed a z'=0.7 ( figure s1 ) (31) . we used this assay pipeline to screen our in-house repurposing library in 384 well plates at a final concentration of 1 µm ( figure 1c ) (32) . 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 s1 ) (8) . using a threshold of <40% infection and >80% viability, as compared to the vehicle control, we identified only six drugs that were antiviral in our primary screen (table s1 ). this included the natural product nanchangmycin, which we previously found in a drug repurposing screen against zika virus (32) . nanchangmycin was broadly antiviral against viruses that enter cells through endocytosis, consistent with the role of endosomal acidification for sars-cov-2 entry in these cells (32) . 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 1d ). 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-2 and monitored infection by microscopy. we initially tested a549, calu-1, huh7, huh7.5, hepg2, hacat, imr90, nci-h292, cfbe41o, and u2os cells. we detected less than 1% infection of a549, calu-1, huh7, hepg2, hacat, imr90, nci-h292, cfbe41o, and u2os cells (data not shown). interestingly, while huh7 were largely non-permissive, the derivative cell line huh7.5 cells was permissive to sars-cov-2 (fig 2a) . huh7.5 cells are defective in innate immune signaling (rig-i) and are known to be more permissive to many viruses, including hepatitis c virus (15) . remdesivir and hydroxylchloroquine were antiviral against sars-cov-2 in huh7.5 cells with ic50s that were greater than 10-fold lower than those observed in vero cells (fig 2b) . we also found that nanchangmycin was antiviral against sars-cov-2 in huh7.5 cells (fig s2) .these observations suggest that huh7.5 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 huh7.5 cells using remdesivir and observed a z'=0.61 ( fig s3) (31) . we screened our repurposing library at 500nm quantifying both the percentage of infected cells as well as cell number to exclude toxic compounds (fig 2c) . we found 33 drugs had antiviral activity in the absence of cytotoxicity (<40% infection, >80% viability, as compared to vehicle control) (table s2 ). this included three of the six drugs identified in vero cells: z-fa-fmk, y-320 and salinomycin. we repurchased powders for 32 drugs and tested their activity in dose-response assays in huh7.5 cells against sars-cov-2. 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 (8) . of those tested, 23 drugs showed activity and fell into diverse classes (fig 2d) . dose-response curves are shown for 23 candidates and the ic50s and cc50s were calculated (fig 2e) . the selectivity index (si, ratio between antiviral and cytotoxicity potencies) was calculated and the 23 candidates were antiviral with si>3 ( figure 2e , table s3 ). dose-responses curves for the other candidates that did not validate in huh7.5 cells are shown in fig s4. 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 (9, 23, 24) . in total, we identified three drugs as antiviral in both screens. we performed dose-responses in vero cells against the candidates from the huh7.5 screen. we found that 5 additional compounds were antiviral in vero cells with a si>3, azd8055, bix01294, ebastine, mg-132, and wye-125132, albeit at higher concentrations ( figure s5 ). however, the majority of the antivirals that were validated in huh7.5 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 a549, calu-1, nci-h292, cfbe41o). however, we found that calu-3 cells, that have been shown to be permissive for many coronaviruses including sars-cov-2, were highly readily infected (fig 3a) (14, 26, 33) . we optimized assays using calu-3 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-3 cells (fig 3b) . 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-2 (fig 3c) , while these compounds are antiviral in both vero cells and huh7.5 cells (fig 3d) . this suggests that there are major differences in the requirement for endosomal acidification during infection of sars-cov-2 in lung epithelial cells. endosomal acidification is thought to be required for sars-cov-2 entry to maintain the low ph necessary for endosomal cysteine protease activity required for priming spike for membrane fusion (26) . consistent with the requirement for acidification in vero and huh7.5, the cathepsin inhibitor z-fa-fmk emerged as antiviral in both cell types (fig 1d, fig 2e) . we tested z-fa-fmk in calu-3 cells and found that it had no antiviral activity (fig 3d) , consistent with a lack of a requirement for endosomal acidification. recent studies found the plasma membraneassociated serine protease, tmprss2, can prime the viral glycoprotein for entry in lung epithelial cells (26) . therefore, we tested the role of tmprss2 by treating cells with the inhibitor camostat. we found that camostat was antiviral in calu-3 cells but had no activity in either vero or huh7.5 cells (figure 3e -f) (26) . moreover, the main endosomal kinase phosphatidylinositiol-3-phosphate/phosphatidylinositol 5-kinase, pikfyve, promotes internalization of diverse viruses and was recently shown to impact entry of coronaviruses including sars-cov-2 in hela cells (34) . using the pikfyve inhibitor apilimod, we found that pikfyve promotes infection of sars-cov-2 in huh7.5 and vero cells, with little importance calu-3 cells ( fig s6) . these data suggest that the entry pathway used by sars-cov-2 is cell-type specific. to determine which of the 23 antiviral candidates validated in huh7.5 cells also had antiviral activity in calu-3 cells we performed dose-response studies. we found that 9 drugs were antiviral against sars-cov-2 in calu-3 cells with a selectivity index greater than 2 (fig 4) . these include: two drugs with unclear targets (salinomycin, y-320), kinase inhibitors (azd8055, bemcentinib, dacomitinib, wye-125132), histamine receptor inhibitor (ebastine), iron chelator dp44mt, 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-3 with a si<3 are shown in fig s7. the full table of candidates from the huh7.5 screen with ic50, cc50 and si are shown in figure s8 . cyclosporine is an fda approved generic drug that is readily available and showed a submicromolar ic50 with high selectivity in both huh7.5 and calu-3 cells (fig 3, fig 4, fig s8) . 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) (35) (36) (37) . inhibition of this pathway in t cells is used as an immunosuppressant (38) . cyclosporins have been shown to have antiviral activity against a wide variety of viruses, including other coronaviruses (39) (40) (41) (42) (43) (44) (45) (46) (47) (48) (49) (50) (51) (52) . the activity of cyclosporine against previously studied coronaviruses is cyclophilin-dependent and independent of calcineurin (50, 51) . 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 (53). we found that isocyclosporin a, cyclosporin a, cyclosporin b, and cyclosporin c were active with increasing ic50s (fig 5a-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 (53, 54) . cyclosporin h has a log reduction in activity compared to cyclosporine, but retains some activity. psc 833 is a non-immunosuppressant derivative of cyclosporine that does not inhibit calcineurin, has a similar activity to cyclosporin c(55). tmn355 is a cyclophilin a inhibitor that is 27 times more potent than cyclosporine a in inhibiting the prolyl isomerase activity of cyclophilin a (56) . 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 5d) . in addition, nim811 is another non-immunosuppressive cyclosporine derivative of cyclosporine, and we found that it is potently antiviral (fig 5e) , 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 s9 ). to further assess the mechanism by which cyclosporine is antiviral, we tested fk506, an inhibitor of calcineurin. fk506 binds the related immunophilin fkbp, rather than cyclophilin a, to block the phosphatase activity of calcineurin, and thus is also a potent immunosuppressant (57) . we found that fk506 has no activity against sars-cov-2 (fig 5a-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 (58) . we found that the nfat inhibitor had no effect on infection (fig 5a-c) . altogether, we found that cyclosporins are potent antivirals against sars-cov-2 in lung epithelial cells, and that this activity is independent of calcineurin and nfat. the emergence of sars-cov-2 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 (59) . remdesivir was developed against the ebola virus rna-dependent rna polymerase, and was also found to have robust activity against sars-cov-2 (8) . importantly, we found that remdesivir is active against sars-cov-2 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-2 (8, 9) . 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 (10) . to determine if there are additional drugs that are active against sars-cov-2 in vitro, we screened a repurposing library that includes ~1000 fda approved drugs and ~1000 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-1 trials (60, 61) . initial screens in vero cells yielded few active drugs, leading us to pursue a screen in human huh7.5 cells, a transformed hepatocyte line deficient in innate immune signaling. using this model system, we identified 33 drugs, and validated 23 with dose-response assays. this includes many drugs that were previously shown to have activity against other coronaviruses (tetrandrine, cepharanthine, cyclosporine, alixostatin, mg132, salinomycin), and sars-cov-2 (salinomycin, tetrandrine, cepharanthine, cyclosporine, ebastine) (13, 40, 50, 54, (62) (63) (64) (65) (66) (67) (68) . the 23 drugs fall into distinct classes and most have known targets. however, two drugs that were active across cell types, salinomycin and y-320, 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 (13, (68) (69) (70) . salinomycin was also identified in a vero cell screen (13) . mechanistically, some studies have suggested that salinomycin is an ionophore that can attenuate viral entry by disrupting the acidification of the endosome (71) . other studies have implicated salinomycin in er stress (72) . studies in mice have shown antiviral activity against influenza (71) . salinomycin has also been characterized as an activator of autophagy, which may influence sars-cov-2 infection (73, 74) (75). y-320 is a phenylpyrazoleanilide immunomodulatory agent that has been shown to inhibit il-17 production by t cells and has activity in monkeys (76) . interestingly, treatment with y-320 is associated with decreased il-6 production, a cytokine that is thought to be highly expressed in sars-cov-2 infection (76) (77) (78) . however, it is unclear how y-320 could attenuate sars-cov-2 in non-immune cells. ebastine is a potent h1-histamine receptor antagonist, used for allergic disorders outside of the us, particularly in asia (79). we found that ebastine is antiviral in all three cell types, although 10-fold less active in vero cells (13) . ebastine is orally available with few side effects and there are there clinical trials underway in china testing whether ebastine can impact covid-19 outcomes (80) . since other h1-histamine receptor antagonists were not active, it is unclear why this particular agent is more effective at inhibiting sars-cov-2 infection. interestingly, ebastine and its active metabolite, carebastine, are reported to inhibit expression of il-6, while many other h1-histamine receptor antagonists do not (81, 82) . we also identified 6 protease inhibitors as antiviral in huh7.5 cells. two cysteine protease inhibitors, z-fa-fmk and mg-132, had activity in both vero and huh7.5 cells. none of the protease inhibitors were active in calu-3 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-2 entry in cells where endosomal proteases are required for spike cleavage, and thus we observe no requirement in calu-3 cells where tmprss2 is required for infection (16, 19, (26) (27) (28) .this has important implications in diverse sars-cov-2 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 g9a as antiviral in huh7.5 cells. however, these drugs were not active in calu-3 cells, suggesting that there are cell type specific requirements. am1241 is a selective cannabinoid cb2 receptor agonist that we found was antiviral in huh7.5 cells. gw842166x, another cb2 agonist that has a 10-fold higher ec50, was not active. moreover, dose-response studied found that am1241 is not active in either vero or calu-3 cells. cepharanthine and tetrandrine are both bis-benzylisoquinoline alkaloids produced as natural products from herbal plants (83) . 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 (84, 85) . multiple studies have suggested that tetrandrine has antiviral activity, including against dengue virus and herpes simplex 1 virus (86, 87) . 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 (88) . currently, there is an ongoing clinical trial using tetrandrine in covid-19 patients to improve pulmonary function (80) . 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 (89) . both cepharanthine and tetrandrine were previously shown to have antiviral activity against the human coronavirus oc43 and in recent studies on sars-cov-2 in vero cell screens (13, 62, 63) . while both of these molecules were antiviral in our huh7.5 screen, neither were active in calu-3 cells. this may suggest that they are modulating endosomal entry pathways. we identified few metabolic regulators. dp44mt is a potent iron chelator that we found to be antiviral against sars-cov-2 in huh 7.5 and calu-3 cells (56) . a clinical trial with the iron chelator deferoxamine is underway (nct04333550). 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-2. frax486 is a p21activated kinase (pak) inhibitor that is antiviral in huh7.5 cells, but only modestly impacted infection of calu-3 cells (90) . other pak inhibitors were not identified in our screens. pak is required for entry by many viruses(91). pd0166285 is a potent wee1 and chk1 inhibitor that is antiviral in huh7.5 cells, but shows strong toxicity in calu-3 cells (92). we also found three mtor inhibitors, azd8055, pf-04691502, and wye-125132 are antiviral against sars-cov-2 in huh-7 and calu-3 cells. these are highly potent atp competitive mtor inhibitors that target both torc1 and torc2. in our library, none of the rapamycin analogs that selectively inhibit mtorc1 were active. we also identified two potent selective and irreversible inhibitors of egfr, dacomitinib and naquotinib. the other 44 egfr inhibitors showed no activity in huh7.5 cells. importantly, dacomitinib is a potent antiviral in calu-3 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-2 (93-97). 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) (35) (36) (37) (38) 41) . inhibition of this pathway in t cells is used as an immunosuppressant. we found that cyclosporine is active in both huh7.5 and calu-3 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-2 (13) . cyclophilin a is a ubiquitously expressed peptidyl-prolyl cis-trans isomerase (102). cyclophilin a and other cyclophilins have chaperone-like activity and take part in protein-folding processes (103) . cyclophilin a has been shown to be an important cellular factor that facilitated many diverse viral infections. this includes human immunodeficiency virus type 1 (hiv-1), 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) (41-49, 104, 105) . the coronaviruses hcov-229e, hcov-nl63, fpiv, mouse hepatitis virus (mhv), avian infectious bronchitis virus, and sars-cov have been found to be attenuated by cyclosporin a (40, 64, 106) . 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 (54, 105), but cypa and cypb were found to be required for fcov replication (106) . for hcov-nl63, and hcov-229e, cyclophilin a is required for infection in caco-2 cells (50) and huh-7.5 cells respectively (51, 52) . 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 tmn355 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 fk506, 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-2 in lung epithelial cells, and that this activity is independent of calcineurins. nim811 is a cyclophilin inhibitor independent of calcineurin, and we found that this is highly active in huh7.5 cells, further suggesting that cyclophilin is required for sars-covo-2 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-2 in diverse infected tissues in vivo. one approach would be to use cyclophilin inhibitors that do not have immunosuppressive activity such as nim811 or others that have been tested for hcv infection (alisporivir (debio-025) and scy-635) (107) or for hiv infection (nim818) (108) (54) . another possibility is to use cyclophilin inhibitors that also target calcineurin (eg. cyclosporine). one of the major complications of covid-19 is the hyper-inflammatory response and cytokine storm associated with increased immune activation. to prevent hyper-activation, there has been interest in treating covid-19 patients with immunosuppressants (109). there are ongoing trials for a variety of agents including anti-il6 and jak inhibitors, two clinical trials using sirolimus, the fda approved mtor inhibitor, which selectively inhibits mtorc1. 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 (110) . therefore, it may be useful to implement clinical trials using cyclosporin a as an immunosuppressant as it would potentially ameliorate symptoms by two mechanisms (111) . 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-2 (https://www.mmv.org/mmv-open/covid-box). we tested >60 of the 80 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 2 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 9 drugs are antiviral in lung epithelial cells, 7 have been used in humans, 3 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-19 patients, to inform future treatment strategies. vero e6 cells and vero ccl81 were obtained from atcc and were cultured in dmem, supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) penicillin/streptomycin, 1% (v/v) l-glutamax and were maintained at 37°c and 5% co2. huh7.5 cells were obtained from c. rice (rockefeller) and cultured in dmem, supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) penicillin/streptomycin, 1% (v/v) l-glutamine, and were maintained at 37°c and 5% co2. calu-3 cells (htb-55) were obtained from atcc and cultured in mem, supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) penicillin/streptomycin, 1% (v/v) l-glutamine, and were maintained at 37°c and 5% co2. sars-cov-2 was obtained from bei (wa-1 strain). stocks were prepared by infection of vero e6 cells in 2% serum plus 10mm hepes for five days, freezethawed, and clarified by centrifugation (po). titer of stock was determined by plaque assay using vero e6 cells and were 1x10 7 pfu/ml and 1.5x10 6 tcid50/ml (6) . this seed stock was amplified in vero ccl81 (p1) at 1.5x10 6 tcid50/ml. all work with infectious virus was performed in a biosafety level 3 laboratory and approved by the institutional biosafety committee and environmental health and safety. infections: cells were plated in 384 well plates (20µl/well) 3,000 cells per well for vero, 3,000 cells per well huh7.5, 7,500 cells per well calu-3. the next day, 50nl 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-2 (vero, moi=1; huh7.5 moi=1; calu-3 moi=0.5) cells were fixed (30hpi vero and huh7,5, 48hpi calu-3) in 4% formaldehyde/pbs for 15min at room temperature and then washed three times with pbst. cells were blocked (2% bsa/pbst) for 60 minutes and incubated in primary antibody (anti-dsrna j2) overnight at 4c. cells were washed 3x in pbst and incubated in secondary (anti-mouse alexa 488 and hoescht 33342) for 1h at room temperature. cells were washed 3x in pbst and imaged using imagxpress micro using a 10x objective. four sites per well were captured. the total number of cells and the number of infected cells were measured using metaxpress 5.3.3 cell scoring module, and the percentage of infected cells was calculated. the aggregated infection of the dmso and remdesivir control wells (n=16) 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 )*100] and z-score [z= (%infection sample -average %infection dmso ) / standard deviation %infection dmso ]in spotfire (perkinelmer). candidate hits were selected as compounds with poc<40% and viability >80%, compared to vehicle control. candidate drugs were repurchased as powders from selleckchem, medchemexpress, and medkoo and suspended in dmso. drugs were arrayed in 8-pt dose-response in 384 well plates. infections were performed using screening conditions. dmso (n=32) and 10 µm remdesivir (n=16) 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 8) was performed on poc infection and cell viability using log10 transformed concentration values to calculate ic50 values for infection and cc50 values for cell viability for each drug/cell line combination. selectivity index (si) was calculated as a ratio of drug's cc50 and ic50 values (si = cc50/ic50). rt-qpcr: huh7.5 (750,000 cells/well) or calu-3 cells (750,000 cells/well) were plated in 6 well plates. the next day, drugs were added and one hour later infected with sars-cov-2 (moi=0.5). total rna was purified using trizol (invitrogen) followed by rna clean and concentrate kit (zymo researc) 24 hpi for huh7.5 or 48 hpi for calu-3. 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-2 (wuhan v1, nsp14) and sybr green master mix (applied biosystems) were used to amplify viral rna and 18s rrna primers were used to amplify cellular rna using the quantstudio 6 flex rt-pcr system (applied biosystems). relative quantities of viral and cellular rna were calculated using the standard curve method (112) . viral rna was normalized to 18s rna for each sample (wuhan v1/18s). wuhan-v1_forward 18s rrna_forward 5'-aacccgttgaaccccatt-3' 18s rrna reverse 5'-ccatccaatcggtagtagcg-3' we thank s. weiss and y. li for sharing sars-related coronavirus 2, isolate usa-wa1/2020 (obtained from the centers for disease control and bei resources). we thank bei resources for quantitative sars-cov-2 rna. we thank m. diamond and s. hensley for providing anti-spike antibody (cr3022), c. coyne for j2 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 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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: 2009-07-02 journal: plos one doi: 10.1371/journal.pone.0006130 sha: doc_id: 352511 cord_uid: gkm7i62s 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 veroand ck-adapted strains showed efficient induction of membrane fusion. however, expression of s protein cloned from the third passage of ibv in chicken embryo (ep3) did not show apparent syncytia formation. by construction of chimeric s constructs and site-directed mutagenesis, a point mutation (l857-f) at amino acid position 857 in the heptad repeat 1 region of s protein was shown to be responsible for its acquisition of the cell–cell fusion activity. furthermore, a g405-d point mutation in the s1 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 l857 back to the s gene of vero-adapted ibv allowed recovery of variants that contain the introduced l857. however, compensatory mutations in s1 and some distant regions of s2 were required for restoration of the cell–cell fusion activity of s protein carrying l857 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 [1] . 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 [2] . 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 [3, 4] . cross-species transmission was also observed in coronavirus transmissible gastroenteritis virus (tgev) and human coronavirus oc43 [5] [6] [7] . 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 [8, 9] and the african green monkey kidney cell line vero cell [8] [9] [10] [11] [12] [13] . furthermore, the veroadapted ibv is able to infect cultured human and animal cell lines [14, 15] . in a previous report, a total of 49 amino acid substitutions was found during adaptation of ibv from chicken embryo (ep3) to vero cells (p65) [10] [11] [12] . among them, 26 amino acid substitutions are in the s protein [10] . in this study, expression of s protein cloned from ibv strains ep3, ck, passage 7 (p7) and p65 of vero-adapted ibv showed induction of cell-cell fusion by s(ck), s(p7) and s(p65) constructs. however, no formation of syncytial cells was observed in cells expressing s(ep3). construction of chimeric s constructs and site-directed mutagenesis studies identified a leucine to phenylalanine substitution at the amino acid position 857 in the heptad repeat 1 region (l857-f) that confers the non-fusogenic s protein to fusogenic. re-introduction of the f857-l mutation back to the genome of vero-adapted ibv (p65) showed rescue of virus containing the f857-l mutation. however, compensatory mutations occurred in the s1 region that could rescue the cell-cell fusion activity of s constructs carrying the f857-l mutation. cells were maintained in dmem supplemented with 10% newborn calf serum. the vero-adapted ibv and recombinant vaccinia/t7 virus was propagated and titrated on vero cells. virus stocks were kept at 280uc until use. cell monolayers grown on 4 well slide chambers were infected with vaccinia/t7 virus for 1 hour followed by transfection of indicated plasmids using the effectene transfection kit (qiagen). at 12 hours post-transfection, cells were washed with phosphate buffered saline (pbs) supplemented with 10% normal goat serum, fixed with 4% paraformaldehyde in pbs for 15 minutes, and permeabilized with 0.2% triton x-100. 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 12 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 12 hours post transfection. cells were washed once with pbs, resuspended in blocking buffer containing 20% fbs and 1% bsa in pbs, and incubated on ice for 30 minutes. subsequently, cells were incubated with 0.1% saponin in facs washing buffer containing 2.5% fbs and 0.05% sodium azide in pbs for 10 minutes at room temperature when required. immunofluorescent staining was carried out with 1:100 diluted rabbit anti-ibv s polyclonal antibodies, and 1:20 diluted fitc-conjugated swine anti-rabbit antibody (dako). after washing two times with the facs washing buffer, cells were fixed with 1% 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 p65 of vero-adapted ibv was previously reported [16, 17] . the f857-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 t7 kit (ambion) according to the manufacturer's instructions with certain modifications. briefly, 30 ml of transcription reaction with a 1:1 ratio of gtp to cap analog were sequentially incubated at 40.5uc for 25 minutes, 37.5uc for 50 minutes; 40.5uc for 25 minutes, and 37.5uc for 20 minutes. the transcripts were extracted with phenol/chloroform. vero cells were grown to 90% confluence, trypsinized, washed twice with cold pbs, and resuspended in pbs. rna transcripts were added to 400 ml of vero cell suspension in an electroporation cuvette, and one electrical pulse at 450 v, 50 mf was given using a bio-rad gene pulser ii electroporator. the transfected vero cells were cultured overnight in 1% fbs-containing mem in a 60 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 pkt0 vector [18] . chimeric s constructs were made by overlapping pcr [19] . 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 ep3 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 [10] . sequence comparison of two s protein constructs, s(ep3) and s(ck), cloned from ep3 and ck-adapted ibv strains, respectively, showed amino acid substitutions at 31 positions (fig. 1a) . the cell-cell fusion activity of these two s constructs was analyzed by transfection into vero cells using the vaccinia/t7 recombinant virus system. western blot analysis showed the presence of major forms of s protein, including the 180-kda glycosylated (s*) and 130-kda unglycosylated full-length s (s) and the cleaved s1 and s2 species (s1/s2) in cells expressing the two constructs (fig. 2a, lanes 2 and 3) . it was noted that the expression level of s(ck) was higher than s(ep3) (fig. 2a) . 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. 2a, lane 1) . immunofluorescent staining of vero cells expressing s(ck) clearly showed syncytia formation at 12 hours post-transfection (fig. 2b , panel s(ck)). however, in vero cells expressing s(ep3), no obvious syncytia was observed (fig. 2b , panel s(ep3)). in the negative control cells, no fusion of the transfected cells was detected (fig. 2b, 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. 2c acquisition of the cell-cell fusion activity by mutation of a conserved leucine residue to phenylalanine (l857-f) in the heptad repeat 1 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 ep3-ck(1) was made by replacing the c-terminal 412 amino acid region of s(ep3) with the corresponding region from s(ck), ep3-ck(2) was made by replacing the cterminal 280 amino acid region of s(ep3) with the corresponding region from s(ck), and ck-ep3 was made by replacing the nterminal 882 amino acid region of s(ep3) with the corresponding region from s(ck) (fig. 3a) . western blot analysis of cells expressing these constructs detected the s1 and s2 species as well as the glycosylated and unglycosylated forms of the full-length s protein (fig. 3b , lanes [3] [4] [5] . immunofluorescent staining showed cell-cell fusion and syncytia formation in cells expressing both ep3-ck(1) and ck-ep3 (fig. 3c , panels ep3-ck(1) and ck-ep3), but not ep3-ck(2) (fig. 3c , panel ep3-ck (2)). 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 1) of cells expressing s(ep3), and are listed in the order from high to low as follows: ck-ep3.ck = ep3-ck(1)&ep3 = ep3-ck(2) (.indicates the relative activity is within 1 fold higher, and &indicates more than 1 fold higher). these results demonstrate that the region between amino acids 750 and 882 may determine the fusogenic difference between s(ep3) and s(ck). examination of this region showed two amino acid substitutions from s(ep3) to s(ck), i.e. n826 to s and l857 to f (fig. 1b) . to determine which amino acid substitution dictates the fusogenic change, three mutant constructs were made. constructs ck3(s826-n) and ck(f857-l) were made by mutation of the s826 and f857 residues in s(ck) to n and l, respectively (fig. 3a) . construct ep3(l857-f) was made by mutation of the l857 residue in s(ep3) to f (fig. 3a) . western blot analysis of cells expressing these constructs detected the s1 and s2 species as well as the fulllength forms (fig. 3b , lanes 6-8). immunofluorescent staining showed formation of syncytia in cells expressing ck(s826-n) (fig. 3c , panel ck(s826-n), suggesting that mutation of s836 to n did not affect the cell-cell activity of s(ck). cell-cell fusion and syncytia formation were also observed in cells expressing ep3(l857-f) but not ck(f857-l) (fig. 3c , panels ep3(l857-f) and ck(f857-l)), demonstrating that the l857-f mutation introduced into s(ep3) renders the protein fusogenic in cultured cells. on the other hand, mutation of the f857 residue to l totally abolishes the fusion activity of s(ck) (fig. 3c , panel ck(f867-l). the relative cell-cell fusion activities of these s constructs are ck(s826-n).ep3(l857-f)&ep3 = ck(f857-l). these results confirm that s(ck) gains the cell-cell fusion activity by l857-f mutation in the heptad repeat 1 region of the protein. the f857-l substitution was then introduced into the s constructs cloned from vero-adapted ibv (p7) and (p65), respectively, generating s(p7) and s(p65) (fig. 3a) . expression of these constructs showed cell-cell fusion and syncytia formation in cell expressing wild type s(p7) and s(p65), but not the mutant s proteins (fig. 3c , panel p7, p7(f857-l), p65 and p65(f857-l)). the expression levels of both f857-l mutants were lower than the wild type constructs, but no significant difference in the s1/s2 cleavage was observed (fig. 3b, lane 9-12 ). the relative cell-cell fusion activities of these s constructs are p65.p7&ep3 = p7(f857-l) = p65(f857-l). these data indicate that s protein acquires its cell-cell fusion activity by the l857-f mutation during adaptation to both ck and vero cells. further mutations of the l857 residue to other amino acids based on s(ep3) were made. as shown in fig. 3a , the l857 was mutated to y, s, e, i and k, respectively. expression of these mutant constructs showed that mutations of l857 to y and s exhibited similar effect on cell-cell fusion as the l857-f mutant. cell-cell fusion and syncytia formation were observed in cells expressing these two mutants (fig. 3c) . however, much less cell-cell fusion and smallersized syncytia were observed in cells expressing l857-e, l857-k and l857-i mutant constructs (fig. 3c) . the relative cell-cell fusion activities of these s constructs are ep3(l857-y).ep3(l857-s).ep3(l857-e).ep3(l857-i) = ep3(l857-k).ep3. introduction of the f857-l substitution back to the genome of vero-adapted ibv and analysis of its effect on viral infectivity in cultured cells the f857-l mutation was then introduced back to the genome of vero-adapted ibv by using an infectious clone system based on p65 [16, 17] 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 3 days post-electroporation, syncytia formation the recombinant wild type and mutant viruses (p0) were recovered from the culture media at 3 and 6 days post-electroporation, respectively, and further propagated on vero cells for 5 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, 10 bacterial clones were randomly chosen from p0, and the complete nucleotide sequence of the s gene was determined to confirm if the recovered virus maintains the f857-l substitution. as shown in table 1, l857 was found in all 10 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 1) . among them, two clones contain a t773-s substitution (flv1), one contains an i769-v substitution (flv2), and two contain q523-l and i769-v substitutions (flv3) ( table 1 ). these results demonstrate that the recovered fl mutant virus from p0 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 p1, p3 and p5 was performed. in the four clones chosen from p1, a homogenous s gene with both q523-l and i769-v (flv3) mutations was found (table 1) . subsequent sequencing of clones derived from p3 and p5 each showed that six out of 10 clones from p3 and two out of 10 clones from p5 are flv3 (table 1 ). the dominant clones contain an additional proline to serine substitution at amino acid position 327 (flv4) ( table 1) . the recovered viruses were then plaque-purified. compared to wild type ibv, ribv showed similar growth kinetics in vero cells (fig. 4b) , but formed slighlty smaller plaques (fig. 4a) with lower expression level of s protein (fig. 4c) . a total of 20 mutant viruses was plaque-purified from passages 3 and 5, and the s gene of all purified viruses was shown to share the same sequence as flv4 ( table 1 ). the flv4 mutant virus formed similar-sized plaques as ribv (fig. 4a) with slightly lower expression of s protein (fig. 4c) . interestingly, the mutant virus produced up to 10-fold higher titers of virus, compared to ribv (fig. 4b) . restoration of the cell-cell fusion activity of s(p65) protein carrying the f875-l mutation by compensatory mutations in the s1 region the cell-cell fusion activity of s proteins cloned from the mutant ibv construct fl and the four variants (flv1, flv2, flv3 and flv4) was analyzed by expression in vero cells. once again, expression of these constructs led to the detection of s1 and s2 species as well as the full-length forms (fig. 5a) . higher levels of s protein were detected in cells expressing s(flv3) and s(flv4), comparing to cells expressing the other two s constructs (fig. 5a) . immunofluorescent staining showed the formation of giant syncytia in cells expressing s(flv3) and s(flv4) (fig. 5b , panels s(flv3) and s(flv4)), but much smaller syncytia were observed in cells expressing s(flv2) (fig. 5b, panel s(flv2) ). no obvious cell-cell fusion was observed in cells expressing s(fl) and s(flv1) (fig. 5b , panels s(fl) and s(flv1)). the relative cell-cell fusion activities of these s constructs are flv4.flv3.flv2&ep3 = fl = flv1. 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(flv2) and s(flv3) was only at the 523 th residue, the s(fl(q523-l)) construct was also created and expressed. the results showed that it displayed a similar cell-cell fusion activity as flv2 ( = fl(i769-v)) ( fig. 5a further enhancement of the cell-cell fusion activity of s protein and adaptation of ibv to cell culture by g405-d substitution vero-adapted ibv gradually increased its infectivity in vero cells by serial passages and a significant difference between p7 and p65 was observed [10] . comparison of amino acid sequences between s(p7) and s(p65) revealed a single mutation at the amino acid position 405 (g405-d) in s(p65) (fig. 1a) . to analyze the possibility that the enhanced infectivity of p65 virus is due to the enhanced cell-cell fusion activity of the corresponding s protein, s(p7) and s(p65) constructs were created and expressed in vero cells. efficient induction of cell-cell fusion was observed in cells transfected with both constructs (fig. 6a, panels s(p7) and s(p65) and 6b, lanes 3 and 4) . comparatively, significantly larger syncytia was observed in cells expressing s(p65) construct than in cells expressing s(p7) (fig. 6a) , demonstrating that the additional g405-d mutation in s(p65) may enhance its cell-cell fusion activity. the g405-d mutation was then introduced into s(ep3) and s(ck) and expressed (fig. 6a) , showing that introduction of g405-d mutation into s(ck) drastically enhanced its cell-cell fusion activity (fig. 6b) . interestingly, introduction of the mutation into s(ep3) and expression of the construct in vero cells showed formation of small syncytial cells (fig. 6b) . the relative cell-cell fusion activities of these s constructs are ck(g405-d).p65.ck&p7.ep3(g405-d).ep3. avian coronaviruses have been isolated from chicken, turkey and pheasant and may exist in many other avian species [20] . 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 2003. 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 l857-f mutation in the heptad repeat 1 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 s1 region (g405-d) could enhance this process. as s protein carrying the l857-f mutation is able to induce cell-cell fusion, but losses the activity when f857 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 s1 domain to a host cell receptor [21] [22] [23] [24] [25] . 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 nl63, and aminopeptidase n for human coronavirus 229e and tgev [26] [27] [28] [29] [30] . 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 ep3 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 [31, 32] . the beaudette strain of ibv was also reported to have an additional binding activity to heparin-like structures [33] . 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 s2 region of the protein [34] [35] [36] [37] [38] . in this study, we demonstrate that l857-f substitution in the s2 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 (g405-d) in p65 virus. this mutation, meanwhile, enhances the infectivity of the virus in cultured cells. the enhancement effect by mutations in the s1 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 f857-l mutation. in variants flv3 and flv4, additional amino acid substitutions (q523-l and i769-v) greatly enhanced the cell-cell fusion activity of the l857-containig s protein and the infectivity of the recovered virus. the involvement of residues in the s1 region in the cell-cell fusion activity of s protein was also demonstrated in other coronaviruses [25] . 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 [39] . this correlation was also observed in this study with more wild type and mutant s constructs. based on data generated from adaptation [10] and cell-cell fusion studies presented here, a model of two-step adaptation process is proposed (fig. 7) . in this model, the adaptation was divided into primary and secondary adaptation (fig. 7) . early passages of vero-adapted ibv, including p7, p12, p14 and p20, belong to the primarily adapted group (fig. 7) . other cell cultureadapted strains, including the ck-adapted and beaudette-us strains cac39114 and cac39300 [8, 9] , and the vero-adapted strain aav98206 described by youn et al. [13] , also belong to this group (fig. 7) . late passages of vero-adapted ibv, including p36, p50 and p65, contain the additional g405-d amino acid substitution and belong to the secondarily adapted group (fig. 7) . except in the cell culture-adapted ibv strains, the l857 residue was found to be absolutely conserved in all coronaviruses sequenced so far. as structural information for this ibv s protein is currently lacking [40, 41] , 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 s1 and some distant s2 regions could compensate the effect of f857-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 [42, 43] . 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/t7 expression system. attempts to obtain more rigorous quantitative data were unsuccessful. as shown in this figure 7 . 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 ep3 is dq001338, p7 is dq001337, p65 is dq001339. b the accession no. for this vero-adapted strain is aav98206. c the accession no. for these two strains are cac39114 and cac39300. doi:10.1371/journal.pone.0006130.g007 study, s protein is inefficiently translocated to the cell surface, probably due to the presence of an er retention signal [44] . 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 s1 and some distant s2 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-oc43, 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 oc43 strains replication and morphogenesis of avian coronavirus in vero cells and their inhibition by momensin coronavirus ibv: partial amino terminal sequencing of spike polypeptide s2 identifies the sequence arg-arg-phe-arg-arg at the cleavage site of the spike precursor propolypeptide of ibv strains beaudette and m41 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 3b gene: functional characterization of the 3b 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 5a open reading frame is not essential for replication induction of p53-independent cell cycle arrest at s-and g 2 /m-phase in cells infected with the coronavirus infectious bronchitis virus promotes viral replication identification of two new polypeptides encoded by mrna5 of the coronavirus infectious bronchitis virus an arginine-to-proline mutation in a domain with undefined functions within the helicase protein (nsp13) 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 3c-like proteinase and determination of a new cleavage site membrane association and dimerization of a cysteinerich, 16-kda polypeptide released from the c-terminal region of the coronavirus infectious bronchitis virus 1a 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 12-amino acid stretch in the hypervariable region of the spike protein s1 subunit is critical for cell fusion activity of mouse hepatitis virus aminopeptidase n is a major receptor for the enteropathogenic coronavirus tgev human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry angiotensinconverting enzyme 2 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 229e 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 ceacam1 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 ceacam1 receptor specificity of the virus strain conformational changes in the spike glycoprotein of murine coronavirus are induced at 37uc either by soluble murine ceacam1 receptors or by ph 8 coronavirus spike protein inhibits host cell translation by interaction with eif3f 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-a59 acquired fusion activity of a murine coronavirus mhv-2 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-345689-5ns1onkw 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: 2009-01-30 journal: vaccines for biodefense and emerging and neglected diseases doi: 10.1016/b978-0-12-369408-9.00011-1 sha: doc_id: 345689 cord_uid: 5ns1onkw abstract the world was struck by surprise when a severe acute respiratory syndrome (sars) epidemic started in 2003 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 2003, 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 p2/vero cell p143). 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 3 (bsl3) containment. our first contact with niaid on the possibility of developing an inactivated vaccine occurred in the first quarter of 2003. 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 2002, 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 2003, similar cases were reported in hong kong and sporadically throughout south-east asia and canada. this severe disease, with a mortality of 5 -10%, spread rapidly around the world and in april 2003, 25 countries on 5 different continents had reported cases [world health organization (who), accumulative sars cases]. as a result, the who issued on march 2003 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 8000 sars cases and 774 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 23, and we submitted our project plan and budget by june 16. preliminary work began in july and august, and by the third week of september 2003, we had a fully executed contract. by october 2004, 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 25, 2003, the who and the cdc in the united states (who guidelines, cdc) recommended laboratories and vaccine manufacturers to handle sars-cov specimens using standard bsl3 work practices. it is not exceptional for vaccine manufacturers to work in bsl3 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 bsl3 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 bsl3 facility. since no treatment or vaccine was available against sars, we decided to work in " bsl3 plus " laboratory conditions. essentially our basic level of containment was bsl3 incorporating several bsl4 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 (20 -50 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, 2003 ; normile, 2004 ; orellana, 2004 ) . furthermore, as described below, the decontamination experiments demonstrated that the sars-cov is an extremely resistant virus. for the medical follow-up of the bsl3-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 bsl3 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 c57bl/6j 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., 2003 ) 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., 2002 ) , a live modified virus against canine cov ( pratelli et al., 2004 ) . 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 12 months to make a good manufacturing practice (gmp) clinical lot for phase 1 and 2 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 ph13 or ph13.5, a strong decrease in the viral infectivity titer can be observed. however, the virus is not totally inactivated and even after 6 h of alkaline treatment, infectious particles can still be observed. total inactivation is observed only after 24 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: 35 -42 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 72 and 144 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 (2 ° 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 58 and 68 ° c. to achieve total inactivation of the sars-cov, 1 h heating at 68 ° c and 2 h heating at 58 ° 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 2 h and then transferred into a tank for thermal decontamination at 105 ° c for 30 min. the laboratory facilities, and the equipment that could not be autoclaved, were decontaminated with 2 ° 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 25, 2003, 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 (vvnfl11) 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 7.0 log/ml. indeed, with the sars-cov, we obtained a consistent titer of around 7.3 log 10 tcid 50 /ml. in vero cells, distinct cytopathic effect (cpe) is always observed at day 2 or 3 post-infection. these first characterization was performed using pcr testing. the pcr tests are listed in table 11 .1 . 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 3, 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-1 cells, human diploid mrc-5 cells, and chick embryo fibroblasts (cefs) were inoculated with the crude viral suspension and are observed for induced cpe and/or hemadsorption. cv-1 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-5 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-5, 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: 1/2000, 1/3000, and 1/4000 (v/v). in our experience, a 1/4000 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 1/4000 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 1.5 log 10 tcid 50 / ml. the kinetics of inactivation was performed at 0, 5, 30 min, and 1, 2, 3, 4, 6, 8, and 24 h, and it was shown that inactivation below the limit of detection was obtained after 6 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 7 days of incubation, the cells are trypsinized and cultivated for additional 14 days. at the different stages of amplification, the cells were microscopically observed for cpe and at the end of the incubation (day 21) an immunocolorimetric assay test was performed. our data demonstrated that the virus is fully inactivated by 12 h, not 6 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 1 pfu. based on these data, it was concluded that inactivation with 1/4000 bpl dilution for 12 h fully inactivates the sars-cov batches. to ensure a very large safety margin, we adopted an inactivation period of 24 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., 2003 ; martina et al., 2003 ; ter meulen et al., 2004 ; rowe et al., 2004 ; mcauliffe et al., 2004 ) . different doses of the sars vaccine (6 or 7 log 10 tcid 50 /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 6 or 7 log 10 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 5 or 6 log 10 . based on our experience to date, the inactivated, adjuvanted sars-cov prototype vaccine seems to be a good candidate for further evaluation in phase 1 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 13). 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 2002, the fda adapted the so-called animal rule [see federal register, may 31, 2002 (volume 67, number 105) ]. 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, 1981 ) . 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 1 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 1 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 1 study. typically, this phase 1 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 1, as with other vaccines, a sars vaccine candidate could move forward to phase 2. 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 2 is successful, following a pre-phase 3 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 3 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 1 to phase 3, 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 3 clinical studies the production of a gmp clinical lot of a monovalent, whole, inactivated, aluminum hydroxideadjuvanted sars-cov vaccine took 12 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 bsl3-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: (1) the realization that the animal testing had to be done in bsl3 facilities by bsl3-trained personnel, and (2) 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 bsl3 or bsl4 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/98 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-003284-hjx2d5rq 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 175 of zika virus ns2a protein affects viral rna synthesis and attenuates the virus in vivo date: 2018-10-07 journal: viruses doi: 10.3390/v10100547 sha: doc_id: 3284 cord_uid: hjx2d5rq 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 (a129 mice) of zikv infection. furthermore, using this infectious clone we have generated a mutant zikv containing a single amino acid substitution (a175v) in the ns2a 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 2016 [1, 2] . 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 175 of the ns2a 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 a129 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 a549 (an human adenocarcinomic alveolar epithelial cell line) cells were purchased from the american type culture collection (atcc, ccl-81) and were grown and maintained at 37 • c and 5% co 2 in growth medium, consisting in dulbecco's modified eagle's medium (dmem) supplemented with 5% fetal bovine serum (fbs) (hyclone, thermofisher scientific, madrid, spain), 2 mm l-glutamine (sigma-aldrich, madrid, spain), 1% nonessential amino acids (sigma-aldrich), 100 u/ml penicillin (sigma-aldrich) and 100 µg/ml streptomycin (sigma-aldrich). the recombinant zikv-rgn wt (rzikv-rgn) or ns2a mutant (rzikv-rgn-mns2a) viruses were propagated in vero cells with virus growth medium (dmem supplemented with 2% fbs, 2 mm l-glutamine, 1% nonessential amino acids, 100 u/ml penicillin and 100 µg/ml streptomycin) at 37 • c and 5% co 2 . for virus stocks preparation, 80 to 90% confluent monolayers of vero cells were infected with a multiplicity of infection (moi) of 0.1 plaque forming units (pfu) per cell in virus growth medium and incubated at 37 • c under 5% co 2 . after 3-4 days of infection, the tissue culture supernatants were collected, clarified by centrifugation at 6000× g for 5 min, and stored in small aliquots at −80 • c. the bac plasmid pbelobac11 [40] , 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 [41] that minimize the toxicity problems in the bacteria of exogenous sequences. e. coli dh10b cells (invitrogen, thermofisher scientific) were used to amplify the bac plasmids. electrocompetent dh10b 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 pbelobac11, based on the data of the full-length sequence of the zikv clinical strain rgn [19] deposited in the genbank (accession number ku527068). 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 2015 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 3347, 5969 and 9127, respectively), which are unique in the viral genome ( figure 1a ). after that, four overlapping dna fragments covering the entire viral genome (zikv 1 to zikv 4) and flanked by the appropriate restriction sites, were generated by chemical synthesis (bio basic, inc., toronto, canada) ( figure 1b ). zikv 1 fragment contained the cmv promoter precisely fused to the first 3350 nucleotides of the viral genome flanked at the 5'-end by apali and asci (absent in the viral genome) sites and at the 3'-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 2 (flanked by pmli and afei) and zikv 3 (flanked by afei and bstbi) covered the genomic regions 3346-5972 and 5967-9131, respectively. zikv 4 fragment contained the restriction site bstbi, the last 1683 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 pbelobac11 by sequential cloning of these four overlapping dna fragments. briefly, fragment zikv 1 was digested with apali and bamhi and cloned into pbelobac11 −afei (a pbelobac11 without the afei restriction site) digested with the same enzymes, to generate the intermediate plasmid pbac-zikv1. then, this plasmid was used as the backbone for the sequential cloning of the remaining overlapping dna fragments (zikv 2 to zikv 4) 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 1b) . 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 dh10b (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 6-well plates were grown to 90% confluence in growth medium without antibiotics, and transfected with 4 µg of the bac cdna clone using 12 µl of lipofectamine 2000 (invitrogen, thermofisher scientific), following the manufacturer's specifications. after 6 h of incubation at 37 • c, the transfection medium was replaced with fresh growth medium and the cells incubated at 37 • c. aliquots of the culture supernatants were collected at 24 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 0.01 pfu/cell) were purified through a 20% (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, 0.5 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 5'and 3'-terminal sequences were determined by the rapid amplification of cdna ends (race) using the 5'/3' race second generation kit (roche, basilea, switzerland) with a polya-tail added to the cdna prior to the 3' 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 a175v in the coding region of the ns2a protein (rzikv-rgn-mns2a), total rna was purified from vero cells infected with viruses from passage 1 (p1) to passage 5 (p5) using the rneasy minikit (qiagen), according to the manufacturer's specifications. purified rna (600 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-3414vs viruses 2018, 10, 547 5 of 21 (5'-gaggaatggtgctgcagg-3'), spanning nucleotides 3414 to 3431 of the viral genome, and the reverse primer zikv-4817rs (5'-gcttgacatctccccag-3') , complementary to nucleotides 4817 to 4833 of the viral genome. finally, the amplicons generated covering the region encoding ns2a and ns2b proteins (genomic region 3414-4833) were sequenced by sanger sequencing (macrogen europe, amsterdam, netherlands) using specific oligonucleotides. vero cells seeded into 12-well plates at 80-90% of confluence were infected with 150 µl of serial 10-fold dilutions of the virus in virus growth medium without fbs for 1 h at 37 • c. after viral absorption, the viral inoculum was removed and the cells overlaid with 2 ml of virus growth medium containing 1% deae-dextran (sigma-aldrich) and 0.6% agar noble (difco, thermofisher scientific). after 3-4 days of incubation at 37 • c under 5% co 2 , the cells were fixed with 4% formaldehyde for 1 h at room temperature, the overlaid removed, and the viral plaques visualized by staining with 0.1% crystal violet in 20% methanol or by immunostaining with 1 µg/ml of the pan-flavivirus e protein monoclonal antibody (mab) 4g2 (bei resources; nr-50327) 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 a549 cells seeded into 24-well plates at 90% of confluence were infected with the indicated viruses diluted in virus growth medium without fbs at the specified mois. after 1 h of absorption at 37 • c in 5% co 2 , the virus inoculum was removed, the cell monolayers washed twice with pbs, and 0.5 ml of fresh virus growth medium was added to each well. cells were incubated at 37 • c under 5% co 2 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 100 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 5'-gaagagcatccagccagagaa-3' (spanning nucleotides 1358 to 1378 of the viral genome), the reverse primer 5'-ctgggagccatgaactgaca-3' (complementary to nucleotides 1399 to 1418 of the viral genome), and the probe 5'-fam-tggagtaccggataatg-3iabkfq-3' (covering nucleotides 1381 to 1397 of the viral genome). to normalize for differences in rna sampling, the expression of the histone h2b (reference housekeeping gene) was analyzed using a specific taqman gene expression assay (rh04253068_s1; life technologies, thermofisher scientific). data were acquired with a 7500 real-time pcr system (life technologies, thermofisher scientific) and analyzed with abi prism 7500 software v2.0.6. the relative quantifications were performed using the cycle threshold (2 −∆∆ct ) method [42] . all experiments and data analysis were miqe (minimum information for publication of quantitative real-time pcr experiments) compliant [43] . the expression of zikv e protein was analyzed by indirect immunofluorescence assay (ifa). vero cells grown on coverslips in 24-well plates at 80-90% of confluence were infected with the rescued rzikvs at the indicated mois. at selected time points post-infection, cells were fixed with 4% paraformaldehyde in 250 mm hepes ph 7.4 during 20 min at room temperature and then permeabilized with 0.5% triton x-100 in pbs for 10 min. after that, cells were treated for 1 h at room temperature with blocking solution (10% fbs in pbs) and incubated with 1 µg/ml of the pan-flavivirus e protein mab 4g2 (bei resources; nr-50327) in blocking solution for 2 h at room temperature. after three washed with pbs, cells were incubated at room temperature for 1 h with donkey anti-mouse antibody conjugated to alexa fluor 488 (invitrogen, thermofisher scientific) diluted 1:500 in blocking solution, extensively washed with pbs, and incubated for 10 min with dapi (4',6'-diamidino-2-phenylindole) (sigma-aldrich) diluted 1:200 in pbs for nuclear staining. finally, coverslips were mounted in prolong gold antifade reagent (invitrogen, thermofisher scientific) and analyzed on a leica sp5 confocal microscope. immunofluorescence acquired images were processed and analyzed with imagej 1.52b software [44] . 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 [45] . briefly, 96-well plates were coated with cell lysates from mock-or zikv-infected vero cells and incubated overnight at 4 • c. the coated wells were washed with pbs, blocked with 1% bsa in pbs, and then incubated with two-fold dilutions (starting dilution of 1:50) of mice sera for 1 h at 37 • c. after that, plates were washed with water and incubated with hrp-conjugated goat anti-mouse igg (1:2000; southern biotech, birmingham, al, usa) for 1 h at 37 • c. reactions were developed with tetramethylbenzidine (tmb) substrate (biolegend, san diego, ca, usa) for 10 min at room temperature, quenched with 2 n h 2 so 4 , and read at 450 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-/-) a129 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, a129 mice retain their adaptive immunity and have been successfully used as a suitable model for testing antivirals and vaccines [46] [47] [48] . to evaluate virus pathogenicity, female 4-to-6-week-old a129 mice (n = 5) were first anesthetized i.p. with a mixture of ketamine (100 µg per gram of body weight) and xylazine (20 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-mns2a diluted in pbs in a final volume of 50 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 14 days. mice showing more than 20% 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, 4-to-6-week-old mice (n = 6) were infected as described above and the viral titers in serum were determined at days 2 (n = 3) and 4 (n = 3) by plaque assay and immunostaining using the pan-flavivirus e protein mab 4g2 as indicated before. to evaluate the protection efficacy of the rzikv-rgn-mns2a, female 4-to-6-week-old a129 mice (n = 5) were first anesthetized i.p. as indicated above, and then mock-immunized (pbs) or immunized s.c. in the footpad with 10 5 pfu of rzikv-rgn-mns2a diluted in pbs in a final volume of 50 µl. at 20 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 10 5 pfu of rzikv-rgn and their morbidity and mortality monitored over 14 days as previously described. to determine viral replication, challenged 4-to-6-week-old a129 mice (n = 6) were bleeding at days 2 (n = 3) and 4 (n = 3) post-challenge and viruses 2018, 10, 547 7 of 21 zikv viremia was determined by plaque assay and immunostaining using the pan-flavivirus e protein mab 4g2 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 <0.05 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 50 (mld 50 ). graphpad prism v7.0 software was used for all statistical analysis. all animal protocols were approved by the university of rochester committee of animal resources (protocol number: ucar-2017-005/101851; approval date: 05/05/2017) and complied with the recommendations in the guide for the care and use of laboratory animals of the national research council [49] . to overcome the toxicity problems associated to several flavivirus sequences during its propagation in bacteria, we used the bac plasmid pbelobac11 (a single-copy plasmid derived from the e. coli f-factor) [40] to assemble a zikv infectious cdna clone, based on the genome sequence of the rgn strain of zikv (genbank accession number ku527068) [19] (figure 1 ). 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 2015 [19] , constituting a good candidate to further study zikv pathogenesis. after appropriate selection of unique restriction sites in the zikv-rgn genome ( figure 1a ), four overlapping dna fragments (zikv 1 to zikv 4), spanning the full-length viral genome and flanked for the selected restriction sites, were chemically synthesized, and sequentially cloned into pbelobac11 to generate the infectious cdna clone pbac-zikv-rgn ( figure 1b ). fragment zikv 1 contained the cmv immediate-early promoter to allow the expression of the viral rna in the nucleus by the cellular rna polymerase ii [50] and fragment zikv 4 was flanked at the 3'-end by the hdv ribozyme followed by the bgh termination and polyadenylation sequences to produce synthetic rnas bearing authentic 3'-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 ku527068). finally, to confirm the stability of this synthetic infectious cdna clone in bacteria, the bac clone was passaged in e. coli dh10b 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 2 ), vero cells were transiently transfected with the bac cdna clone using lipofectamine 2000 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 10 7 pfu/ml on day five ( figure 2a ). to further confirm the identity of the rescued virus, vero cells were infected with an moi of 0.5 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 4g2 ( figure 2b ). 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 2 ), vero cells were transiently transfected with the bac cdna clone using lipofectamine 2000 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 10 7 pfu/ml on day five (figure 2a ). to further confirm the identity of the rescued virus, vero cells were infected with an moi of 0.5 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 4g2 ( figure 2b ). 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 a549 cells, reaching peak titers of approximately 10 7 and 10 6 pfu/ml at 48 hpi, respectively ( figure 3a ). in addition, the rescued virus generated homogeneous plaques of about 2 mm in size after four days of infection in vero cells ( figure 3b ). 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 a549 cells, reaching peak titers of approximately 10 7 and 10 6 pfu/ml at 48 hpi, respectively ( figure 3a ). in addition, the rescued virus generated homogeneous plaques of about 2 mm in size after four days of infection in vero cells ( figure 3b ). 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 a549 cells, reaching peak titers of approximately 10 7 and 10 6 pfu/ml at 48 hpi, respectively ( figure 3a ). in addition, the rescued virus generated homogeneous plaques of about 2 mm in size after four days of infection in vero cells ( figure 3b ). 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 4) , groups of five female 4-to-6-week-old a129 mice (ifnr-/-) were inoculated s.c. in the footpad with pbs (as negative control) or with different doses of rzikv-rgn (10 3 , 10 4 and 10 5 pfu per animal) and the morbidity (body weight loss and disease signs) and survival were monitored daily over 14 days ( figure 4a ). as expected, weight loss and survival correlated with the inoculated dose. mice infected with 10 3 pfu did not show disease symptoms, only a slight reduction in body weight was detected on days 8 to 12, and all of them survived. in the case of mice infected with 10 4 pfu, they presented some symptoms of disease (hunching and reduced mobility) and weight loss from days seven to nine (with a maximum of 10% on day nine), but all of them recovered the initial body weight and survived. in contrast, animals infected with 10 5 pfu showed hind limb paralysis, rapidly lost weight, and all of them succumbed to viral infection between days seven and eight post-infection ( figure 4a ). using the reed & muench method we determined that the mld 50 of rzikv-rgn was approximately 5 × 10 4 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 4b ). 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 10 3 or 10 4 pfu, viremia was not detected or only detected in one of the three infected mice, respectively ( figure 4b ). overall, these results indicated that the rzikv-rgn recovered from the infectious clone is virulent in mice but only at high (10 5 pfu) dose. to determine whether the rescued rzikv-rgn was pathogenic in vivo (figure 4) , groups of five female 4-to-6-week-old a129 mice (ifnr-/-) were inoculated s.c. in the footpad with pbs (as negative control) or with different doses of rzikv-rgn (10 3 , 10 4 and 10 5 pfu per animal) and the morbidity (body weight loss and disease signs) and survival were monitored daily over 14 days ( figure 4a ). as expected, weight loss and survival correlated with the inoculated dose. mice infected with 10 3 pfu did not show disease symptoms, only a slight reduction in body weight was detected on days 8 to 12, and all of them survived. in the case of mice infected with 10 4 pfu, they presented some symptoms of disease (hunching and reduced mobility) and weight loss from days seven to nine (with a maximum of 10% on day nine), but all of them recovered the initial body weight and survived. in contrast, animals infected with 10 5 pfu showed hind limb paralysis, rapidly lost weight, and all of them succumbed to viral infection between days seven and eight post-infection ( figure 4a ). using the reed & muench method we determined that the mld50 of rzikv-rgn was approximately 5 × 10 4 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 4b ). 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 10 3 or 10 4 pfu, viremia was not detected or only detected in one of the three infected mice, respectively ( figure 4b ). overall, these results indicated that the rzikv-rgn recovered from the infectious clone is virulent in mice but only at high (10 5 pfu) dose. week-old a129 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 14 days. mice that lost more than 20% 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 10 3 and 10 4 are statistically significant when data are compared using the unpaired t test (*, p < 0.05; **, p < 0.01). (b) viral titers in mice sera. female 4-to-6-week-old a129 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 4g2. 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 < 0.01; ***, p < 0.001). &: virus not detected in two mice; nd: virus not detected. the detection limit of the assay (200 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 ns2a protein, which was introduced during the chemical synthesis of fragment zikv 2. this mutation consists in a cytosine-to-thymidine substitution at genomic position 4069, resulting in an alanine-to-valine change in the residue 175 of the ns2a protein (a175v). 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-mns2a was generated by replacing the zikv 2 wt fragment for that containing the ns2a a175v 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-mns2a mutant virus was compared to that of the parental rzikv-rgn virus ( figure 5 ). although the infectious virus was recovered in both cases, virus production was one logarithm lower in the case of the mutant rzikv-rgn-mns2a, reaching maximum titers of 10 6 pfu/ml at seven days post-transfection ( figure 5a ). when the plaque phenotype was analyzed, we found that the plaque size of the mutant rzikv-rgn-mns2a was smaller (more than a 5-fold reduction) than that of the parental rzikv-rgn ( figure 5b ), indicating that the a175v 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 175 of the ns2a protein in more than 700 zikv strains sequences deposited in the database [51] (https://www.viprbrc.org/brc/home.spg?decorator=flavi). this analysis indicated that amino acid a175 is highly conserved, since the 100% of the analyzed zikv sequences contained an alanine residue at this position. to further confirm the effect of the ns2a a175v mutation on virus production, the growth kinetics at high (2 pfu/cell) and low (0.05 pfu/cell) moi of the mutant virus were compared to those of the parental virus ( figure 5c ). 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 5c ). taken into consideration that flavivirus ns2a protein is involved in regulation of rna replication and virus assembly [5] , 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 0.5 pfu/cell was analyzed at 24 and 36 hpi by rt-qpcr using a custom taqman assay specific for zikv-rgn genome ( figure 5d ). at both times, a 5-fold reduction in the levels of viral rna was observed in cells infected with the mutant virus ( figure 5d ), confirming that ns2a a175v 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 5e ). 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 ns2a a175v were detected. collectively, these results indicated that ns2a a175v 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-mns2a in vero cells could result in viral attenuation in vivo, the ability of the mutant virus to induce pathogenesis was analyzed in a129 mice and compared with that of the parental rzikv-rgn ( figure 6 ). to that end, groups of five female 4-to-6-week-old a129 mice were inoculated s.c. in the footpad with 10 5 pfu of either rzikv-rgn or rzikv-rgn-mns2a, or with pbs as a negative control, and the body weight loss and survival were monitored daily over 14 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-mns2a did not presented any clinical signs of infection or weight loss and all of them survived to viral infection ( figure 6a ). 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 6b ). the mutant virus was only detected at day two after infection and at lower titers (approximately 1.5 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-mns2a formed small plaques ( figure 6c ). these results indicated that rzikv-rgn-mns2a 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-mns2a mutant virus. after elucidating that rzikv-rgn-mns2a was attenuated in vivo, its ability to induce protection against a challenge with the parental rzikv-rgn was analyzed (figure 7) . to that end, groups of five female 4-to-6-week-old a129 mice were vaccinated s.c. in the footpad with 10 5 pfu of rzikv-rgn-mns2a 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 (10 5 pfu) of rzikv-rgn and the body weight loss and survival were analyzed daily over 14 figure 6 . pathogenesis of rzikv-rgn-mns2a in a129 mice. (a) weight loss and mortality. female 4-to-6-week-old a129 mice (five mice per group) were mock-infected (pbs) or infected s.c. in the footpad with 10 5 pfu of rzikv-rgn or rzikv-rgn-mns2a, and body weight loss (expressed as the percentage of starting weight, left panel) and survival (right panel) were monitored daily during 14 days. mice that lost more than 20% 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 4-to-6-week-old a129 mice (six mice per group) were infected with 10 5 pfu of rzikv-rgn (wt) or rzikv-rgn-mns2a (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 4g2. 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-mns2a are statistically significant when data are compared using the unpaired t test (***, p < 0.001). nd: virus not detected. the detection limit of the assay (200 pfu/ml) is indicate as a dashed line. (c) plaque phenotype. vero cells at 90% confluence (6-well plate format) were infected with 25 pfu of rzikv-rgn (left) or rzikv-rgn-mns2a (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 4g2. after elucidating that rzikv-rgn-mns2a was attenuated in vivo, its ability to induce protection against a challenge with the parental rzikv-rgn was analyzed (figure 7) . to that end, groups of five female 4-to-6-week-old a129 mice were vaccinated s.c. in the footpad with 10 5 pfu of rzikv-rgn-mns2a 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 (10 5 pfu) of rzikv-rgn and the body weight loss and survival were analyzed daily over 14 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-mns2a did not lose weight and all of them survived the challenge with rzikv-rgn ( figure 7a ), indicating that a single immunization dose with rzikv-rgn-mns2a 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-mns2a ( figure 7b ) and no viremia was detected in sera samples of vaccinated mice at days two and four after challenge ( figure 7c ). figure 7a ), indicating that a single immunization dose with rzikv-rgn-mns2a 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-mns2a ( figure 7b ) and no viremia was detected in sera samples of vaccinated mice at days two and four after challenge ( figure 7c ). once confirmed that rzikv-rgn-mns2a 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 8 ). to this end, both mutant (rzikv-rgn-mns2a) and parental (rzikv-rgn) viruses were passaged five times in vero cells (p1 to p5) and the virus plaque phenotype, growth kinetics and the sequence of ns2a 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 p2 (2% of big plaques), clearly increased at p3 (45% of big plaques) and was complete at p4 ( figure 8a ). after that, we analyzed whether this plaque size reversion of the mutant rzikv-rgn-mns2a correlated with an increase in virus replication to levels of that of the parental virus. to that end, the growth kinetics (moi of 1 pfu/cell) of the mutant virus from p1 and p5 were compared to those of the parental virus. growth curve analysis showed that in contrast to the mutant virus from p1, the virus from p5 replicated to the same levels as the parental virus ( figure 8b ), suggesting that the mutant virus reverted to the wt sequence during its propagation in vero cells. to confirm these observations, the ns2a coding region of mutant viruses from p1 to p5 was amplified by rt-pcr and sequenced. sequence analysis confirmed the reversion of the a175v 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 ns2a residue for virus replication. at 72 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 1 to 5 (p1 to p5) were generated. (a) plaque size. vero cells were infected with the different passages (p1 to p5) of rzikv-rgn or rzikv-rgn-mns2a, and at four days post-infection the viral plaques were visualized by immunostaining using the pan-flavivirus e protein mab 4g2. (b) growth kinetics. vero cells at 90% confluence (24-well plate format; triplicates) were infected (moi of 1 pfu/cell) with p1 and p5 of rzikv-rgn or rzikv-rgn-mns2a 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-mns2a p1 and the experimental samples, rzikv-rgn p1, rzikv-rgn p5 and rzikv-rgn-mns2a p5, are statistically significant when data are compared using the unpaired t test (***, p < 0.001). 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 p2 (2% of big plaques), clearly increased at p3 (45% of big plaques) and was complete at p4 ( figure 8a ). after that, we analyzed whether this plaque size reversion of the mutant rzikv-rgn-mns2a correlated with an increase in virus replication to levels of that of the parental virus. to that end, the growth kinetics (moi of 1 pfu/cell) of the mutant virus from p1 and p5 were compared to those of the parental virus. growth curve analysis showed that in contrast to the mutant virus from p1, the virus from p5 replicated to the same levels as the parental virus ( figure 8b ), suggesting that the mutant virus reverted to the wt sequence during its propagation in vero cells. to confirm these observations, the ns2a coding region of mutant viruses from p1 to p5 was amplified by rt-pcr and sequenced. sequence analysis confirmed the reversion of the a175v 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 ns2a residue for virus replication. moreover, these data also suggest that zikv ns2a 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 [14] [15] [16] [17] [18] [19] , 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 [24] [25] [26] . 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 [29, 30] , insertion of introns to disrupt toxic sequences [31] [32] [33] , mutational silencing of cbps present in the viral genome [37] , in vitro ligation of cdna fragments [27, 28, 34] , the isa method [35, 36] , and the cper approach [38] . 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 [28, 32, 34, 36] . 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 pbelobac11 [40] under the control of the cmv promoter, which allows the expression of the viral rna in the nucleus [50] , and flanked at the 3'-end by the hdv ribozyme and the bgh polyadenylation and termination sequences to produce synthetic rnas bearing authentic 3'-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 a129 mice. a zikv-rgn infectious clone generated using the cper approach has been recently reported [38] . however, in contrast to our results, the rescued virus was asymptomatic and nonlethal in female 8-to-12-week-old a129 mice infected with doses of 10 3 to 10 6 ccid 50 (50% 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 (8-to-12-week-old versus 4-to-6-week-old) or the moi used to infect the mice (10 3 -10 6 ccid 50 versus 10 5 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 [41] . 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 [32, 33, 35, 36, 38] , 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 [52] ; (iii) like other systems based on transfection of dna constructs [32, 33, 35, 36, 38] , 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 [53] [54] [55] [56] ; and (v) the bac approach has been successfully used to engineer infectious clones of other flaviviruses, including denv [39] , and several coronaviruses that contain the largest viral rna genome known and similar toxicity problems to those described for flaviviruses [52, [57] [58] [59] [60] . 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 (a175v) in the viral ns2a 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 ns2a protein could affect other steps in the replication cycle of the virus. flavivirus ns2a protein is a 22-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 [61, 62] , virus assembly [63, 64] , membrane rearrangement [64] , and immunomodulation of innate immune response [65] [66] [67] . by homology with the denv ns2a topology, the zikv ns2a a175v 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 ns2a domain in viral rna synthesis. on the other hand, it is important to note that the ns2a a175v mutation promoted a 5-fold reduction in viral rna synthesis and more than 10-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 ns2a protein is also involved in virus assembly, we cannot discard an additional effect of a175v mutation in zikv assembly. in addition, we have found that the mutant virus was attenuated in a129 mice. this attenuation could be explained as a consequence of the lower rna synthesis of the mutant virus. however, an additional effect of the a175v mutation on the putative immunomodulatory role of ns2a protein [65] [66] [67] , 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 10 5 pfu of the mutant rzikv-rgn-mns2a induced protection against a lethal challenge with the parental rzikv-rgn, suggesting the potential implementation of this ns2a mutant as the base of a live-attenuated vaccine. unfortunately, the mutant rzikv-rgn-mns2a 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 a175 of the ns2a protein among zikv strains highlights the importance of this ns2a residue for virus 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fragments of human dna in escherichia coli using an f-factor-based vector analysis of relative gene expression data using real-time quantitative pcr and the 2(−∆∆ c(t)) method. methods the miqe guidelines: minimum information for publication of quantitative real-time pcr experiments an open-source platform for biological-image analysis canine influenza viruses with modified ns1 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-2 and studies of the role of orf3abc in viral cell tropism recovery of a neurovirulent human coronavirus oc43 from an infectious cdna clone subcellular localization and some biochemical properties of the flavivirus kunjin nonstructural proteins ns2a and ns4a 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 ns2a selectively block production of infectious particles role of nonstructural protein ns2a in flavivirus assembly analysis of adaptive mutations in kunjin virus replicon rna reveals a novel role for the flavivirus nonstructural protein ns2a in inhibition of β interferon promoter-driven transcription a single amino acid substitution in the west nile virus nonstructural protein ns2a 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-299509-7xjdryoq 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 e1-226v isolates date: 2013-08-01 journal: plos one doi: 10.1371/journal.pone.0071047 sha: doc_id: 299509 cord_uid: 7xjdryoq chikungunya virus (chikv) is a mosquito-borne alphavirus that re-emerged in 2004 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 e1-226v chikv isolates, we have constructed a new chikv full-length cdna clone, chikv ls3, 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 ls3-gfp). their characteristics were compared to those of natural strain ita07-ra1, which was isolated during the 2007 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 ita07-ra1 and clinical isolate nl10/152, the synthetic viruses displayed similar sensitivities to several antiviral compounds. 3-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 virusesdo 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 ls3 and ls3-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 2004 and has caused unprecedented outbreaks in asia and africa since 2005. the estimated number of cases exceeds 2 million and over a thousand infected travelers have returned to europe and the usa since 2006 [1, 2] . 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 [3, 4] . a licensed vaccine or specific antiviral therapy are currently not available. chikv is an alphavirus with an 11.7 kb positive-stranded rna genome that contains two open reading frames (orfs). the 59 orf encodes the nonstructural polyproteins p123 and p1234. the latter results from translational read-through of an opal termination codon that is present at the end of the nonstructural protein (nsp) 3 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 nsp2 will ultimately lead to the release of nsp1, nsp2, nsp3, and nsp4. 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 [5] . 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, e3, e2, 6k and e1. 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 [5] [6] [7] . the chikv strains that emerged during the 2005-2006 outbreaks had acquired a mutation (a226v) in the e1 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 [8, 9] . 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 a226v substitution in e1 [10] [11] [12] [13] . 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 [14] . 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 2007 and france in 2010 [15, 16] . 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 37997 strain that was isolated from a mosquito -were all based on clinical isolates from infected humans [17] [18] [19] [20] [21] [22] [23] . 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 [24] . 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 e1-226v chikv strains that were circulating during the 2005-2009 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 ls3 (leiden synthetic 3), and a variant that expresses the egfp reporter gene under control of a duplicated subgenomic promoter (chikv ls3-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 (ita07-ra1) from the 2007 chikv outbreak in italy demonstrated that they have similar characteristics. the sensitivity of ls3 to a number of antiviral compounds was compared to those of ita07-ra1 and clinical isolate nl10/152. all compounds tested had a similar antiviral activity against ls3 and the natural isolates. these experiments also identified 3-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 e6, ae. albopictus c6/36 [25] and 293/ace2 cells [26] were maintained in dulbecco's modified eagle's medium (dmem; lonza), supplemented with 8% fetal calf serum (fcs; paa), 2 mm l-glutamine, 100 iu/ml of penicillin and 100 mg/ml of streptomycin. 293/ace2 cells were grown in the presence of 12 mg/ml blasticidin (paa) and c6/36 medium was supplemented with non-essential amino acids (lonza). bhk-21 cells were cultured in glasgow's modified eagles medium (gibco) supplemented with 7.5% fcs, 10 mm hepes ph 7.4, 8% tryptose phosphate broth (gibco), and antibiotics. the mammalian cell lines were grown at 37uc and c6/36 cells at 30uc in 5% co 2 . chikv strain ita07-ra1 (genbank accession number eu244823) was isolated from ae. albopictus during the 2007 outbreak in ravenna, italy, and was passaged twice on bhk-21 cells. chikv nl10/152 (genbank kc862329) 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 e6 cells at 37uc, typically yielding titers of ,10 7 pfu/ml. infections were performed in eagle's minimal essential medium (emem; lonza) with 25 mm hepes (lonza) supplemented with 2% fcs, l-glutamine, and antibiotics. after 1 h, the inoculum was replaced with fresh culture medium. all procedures with live chikv were performed in a biosafety level 3 facility at the leiden university medical center. a cdna clone of the synthetic chikv strain ls3-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 ls3-gfp were chemically synthesized (geneart, germany). using standard cloning techniques, these fragments were assembled and cloned into the asci-noti sites of vector puc19an, a puc19-derived plasmid in which the original polylinker was replaced by one with asci-ncoi-ecorv-xhoi-noti sites. the resulting plasmid (pchikv-ls3-gfp) contains the genomic cdna of chikv ls3-gfp directly downstream of a phi2.5 promoter and followed by a unique spei linearization site for dna linearization prior to in vitro transcription. the 'wild type' synthetic pchikv-ls3 construct was made by deleting the 920 bp egfp-containing saci fragment from pchikv-ls3-gfp (fig. 1) . a third variant with a duplicated subgenomic promoter and a multiple cloning site behind subgenomic promoter 1 (pchikv-ls3-mcs), which allows the introduction of e.g. a reporter gene, was generated by removing the 737 bp asisi-paci fragment from pchikv-ls3-gfp. the constructs were verified by sequencing. in vitro transcription and rna transfection rna was transcribed from plasmids with the phi2.5 promoter [27] using the ampliscribe t7 high yield transcription kit (epicenter), the m 7 gpppa rna cap structure analog (neb) and 0.7 mg of template dna that had been linearized with spei. after a 3-h reaction at 37uc, template dna was digested with dnasei and rna was purified by precipitation with 7.5 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-21 cells (2610 6 ) were electroporated with 1 mg of rna using program t-20 of the amaxa nucleofector and kit t (lonza) according to the manufacturer's instructions. electroporated cells were plated in 6well clusters and incubated at 37uc 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-39 (gactgca-gatgcccgccatt), at-41 (cgctcggtccagg-caactct), at-43 (cgtggtgtttgccaacaggc), or at-52 (cgccgttttttttttttttttttttttttt). in the second step 4 pcr products were generated using combinations of primers at-38 (atggctgcgtgagacacacg) and at-39, at-40 (tgcacccaagtgtaccacaa) and at-41, at-42 (caggagagtgcatccatggc) and at-43, or at-44 (gaatgcgcgcagatacccgt) and at-52. the resulting rt-pcr products were purified and directly sequenced (50 ng template) using the bigdye terminator cycle sequencing kit v1.1 (applied biosystems) and a 3130 genetic analyzer automatic sequencer (applied biosystems). pcr conditions and primer sequences are available upon request. viral titers were determined by plaque assay on vero e6 cells. six-well clusters containing confluent monolayers of vero e6 cells were incubated with 0.5-ml volumes of 10-fold serial dilutions of chikv-containing samples. after a 1-h incubation at 37uc, the inoculum was replaced with 2 ml of dmem containing 1.2% avicel rc-581 (fmc biopolymer), 2% fcs, 25 mm hepes, and antibiotics. after a 66-h incubation at 36uc, monolayers were fixed with 3.7% formaldehyde in pbs and plaques were visualized by crystal violet staining. for infectious center assays 10-fold serial dilutions of electroporated cells were added to 6-well clusters already containing a monolayer of 1610 6 bhk-21 cells per well. after a 1-h incubation at 37uc, a dmem/avicel overlay was applied and cells were incubated at 37uc for 48 h. plaques were visualized as described above. total rna was isolated from 7610 5 cells by lysis in 0.5 ml of 20 mm tris-hcl (ph 7.4), 100 mm licl, 2 mm edta, 5 mm dtt, 5% (w/v) lithium dodecyl sulfate, and 100 mg/ml proteinase k. after acid phenol (ambion) extraction, rna was precipitated with isopropanol, washed with 75% ethanol, and dissolved in 1 mm sodium citrate (ph 6.4). samples containing rna from 4.7610 4 cells were mixed with 3 volumes of 67% formamide, 23% formaldehyde, 6.7% glycerol, 13 mm mops (ph 7.2), 6.7 mm naac, 2.7 mm edta, 0.07% sds, and 0.03% bromophenol blue. after denaturation for 15 min at 75uc, rna was separated in 1.5% denaturing formaldehyde-agarose gels using the mops buffer system as described [28] . rna molecules were detected by direct hybridization of the dried gel with 32 p-labeled oligonucleotides essentially as described previously [29] . positive-stranded genomic and subgenomic chikv rnas were visualized with probe chikv-hyb4 (59-tgtgggttcggagaatcgtg-gaagagtt-39) that is complementary to the 39 end of the genome. negative-stranded rna was detected with probe chikv-hyb2 (59-aacccatcatggatcctgtgtacgtg-ga-39) that is complementary to the 39 end of the minus strand. 18s ribosomal rna (loading control) was detected with the oligonucleotide probe 59-atgcccccggccgtccctct-39. probes (10 pmol) were labeled with 10 mci [c-32 p]atp (perki-nelmer) in a 1h reaction using 10 u of t4 polynucleotide kinase (invitrogen) in 10 ml of the supplied forward reaction buffer (invitrogen). prehybridization (1 h) and hybridization (overnight) were done at 55uc in 56 sspe (0.9 m nacl, 50 mm nah 2 po 4 , 5 mm edta, ph 7.4), 56 denhardt's solution, 0.05% sds, and 0.1 mg/ml homomix i. hybridized gels were washed twice in 56 sspe with 0.05% sds before they were exposed to storage phosphor screens. after scanning with a typhoon-9410 scanner (ge healthcare), quantification of rna levels was done with quantity one v4.5.1 (biorad) and corrections for loading variations were made based on the quantity of 18s 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 7610 5 cells in 0.5 ml of 46 laemmli sample buffer (100 mm tris-hcl, ph 6.8, 40% glycerol, 8% sds, 40 mm dtt, 0,04 mg/ml bromophenol blue). proteins were separated by sds-page in 12% polyacrylamide gels and were transferred to hybond-lfp membranes (ge healthcare) by semi-dry blotting. after blocking with 1% casein (sigma) in pbs with 0.1% tween-20 (pbst), membranes were incubated overnight with rabbit antisera against chikv nsp1 (raised using the peptide eveprqvtpndhan), nsp4 (raised using the peptide assrsnfeklrgpv) or e2 [30] in pbst with 0.5% 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 cy3-conjugated mousea-biotin (jackson) were used for fluorescent detection of the primary antibodies with a typhoon-9410 scanner (ge healthcare). at various time points post infection approximately 2610 5 chikv-infected or mock-infected 293/ace2 cells in 12-well clusters were incubated with 40 mci of 3 h-uridine in medium and incorporation was allowed to proceed for 60 minutes at 37uc. total rna was isolated and analyzed in a denaturing agarose gel as described above. for fluorographic detection of 3 h-labeled rna, the gel was soaked in methanol for 1 hour (one change) and then incubated with 3% 2,5-diphenyloxazole in methanol for at least 3 hours. after incubation in milliq for 30 minutes, the gel was dried and a fuji rx film was placed on top. films were developed after a 1-4 day exposure at 280uc and scanned with a biorad gs-800 densitometer. to check equal sample loading, the gel was hybridized with a 32 p-labeled 18s ribosomal rna-specific probe as described above. in addition, incorporation of 3 h-uridine into rna was quantified by analyzing 2-ml samples of isolated total rna with a liquid scintillation counter (beckman ls 6500 ic). in control samples, cellular transcription was inhibited by adding actinomycin d (sigma) to a final concentration of 5 mg/ml. at various time points post infection approximately 2610 5 chikv-infected or mock-infected 293/ace2 cells in 12-well clusters were starved in dmem lacking l-methionine and lcysteine (invitrogen) for 30 min., and subsequently incubated with 44 mci easytag express 35 s protein labeling mix (perkinelmer) for 30 min. total protein samples were analyzed by sds-page as described above. gels were stained with coomassie to check equal sample loading and 35 s-labeled proteins were detected by drying the gels and exposing them to a storage phosphor screen, which was scanned 1-2 days later with a typhoon-9410 scanner (ge healthcare). chikv-or mock-infected vero e6 cells grown on coverslips were fixed with 3% paraformaldehyde in pbs. after quenching with 10 mm glycine in pbs, cells were permeabilized with 0.1% triton in pbs for 10 min. and coverslips were incubated with primary antibodies diluted in pbs with 5% fcs for 1 h. doublestranded rna was detected with a 1:200 dilution of mouse monoclonal antibody j2 (english & scientific consulting). chikv e2 was visualized with a 1:8000 dilution of a polyclonal rabbit antiserum [30] . detection of primary antibodies was done with donkey-a-mouse-cy3, goat-a-rabbit-cy3 or goat-a-rabbit-alexa488 (1:500; jackson). nuclei were stained with hoechst 33342. the coverslips were mounted with prolong (invitrogen) and analyzed using an axioskop2 mot plus fluorescence microscope with axiocam hrc camera and axiovision software (zeiss). mouse monoclonal antibodies raised against chikv particles of strain ita07-ra1 (izsler, brescia, italy) were heatinactivated for 30 min. at 56uc. two-fold serial dilutions of the neutralizing monoclonal antibody 1h7 and non-neutralizing control antibody 3h9 [31] were incubated with an equal volume of medium containing 100 pfu of chikv. these mixtures were incubated for 60 min. at 37uc and transferred to 96-well clusters containing 2610 4 vero e6 cells per well. after incubation at 37uc for 2 days, the wells were fixed with 3.7% formaldehyde and cpe was detected by staining with crystal violet. chloroquine, 6-aza-uridine and ribavirin were dissolved in pbs. cyclosporin a and 3-deaza-adenosine were dissolved in dmso. mycophenolic acid was dissolved in ethanol. all compounds were obtained from sigma. for cpe reduction assays, 96-well clusters with ,1610 4 vero e6 cells/well were incubated with 50 pfu of virus per well, corresponding to a multiplicity of infection (moi) of 0.005, and 2-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 96h aqueous non-radioactive cell proliferation assay (promega). cpe reduction experiments with ribavirin were done with bhk-21 cells in a similar way, except that viability was assessed 2 days post infection. for egfp reporter gene assays, ,1610 4 vero e6 cells/well in black 96-well plates were infected with chikv ls3-gfp at an moi of 0.05. after a 42-h incubation in medium containing the compound, the cells were fixed with 3% paraformaldehyde in pbs. egfp expression was quantified using a berthold mithras lb 940 plate reader, with excitation and emission wavelengths of 485 and 535 nm, respectively. the fluorescence in wells containing mock-infected cells was used to correct for background signal. ic 50 and cc 50 values were calculated with graphpad prism 5 using the nonlinear regression model. all animal experiments described in this paper were carried out in the bsl3 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 c57bl/6 mice were injected intraperitoneally with 100 tcid 50 of chikv s27, chikv ls3 or chikv ls3-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 7500 detection instrument. the primers and probe used for chikv rna quantification were essentially as described [32] 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 50 equivalents per gram of brain tissue. all animal experiments described in this paper were carried out in the bsl3 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. emc2838/122-12-29). the genbank accession numbers for the full-length cdna clones pchikv-ls3, pchikv-ls3-gfp and pchikv-ls3-mcs are jx911334, jx911335, and jx911336 respectively. the genbank accession numbers for the genomic rna sequences of chikv ls3, ls3-gfp, lcs3-mcs and nl10/152 are kc149888, kc149887, kc149889, and kc862329, respectively. the complete genomes of the 13 chikv strains carrying the e1-a226v mutation ( table 1 ) that were available in genbank at the time of in silico design (november 2009) were aligned using mafft [33] and the resulting consensus sequence formed the basis for the synthetic full-length cdna clones. a 40 nucleotide polya tail was added to the 39 end of the consensus sequence and an a7435g 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 ls3 (genbank accession kc149888). variants containing a duplicated subgenomic promoter and a multiple cloning site (chikv ls3-mcs; genbank kc149889) or an egfp reporter gene (chikv ls3-gfp; genbank kc149887) 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 [18] . the chikv cdnas were placed downstream of a phi2.5 t7 promoter, and a unique spei site for linearization prior to in vitro transcription directly followed the polya tail. the phi2.5 promoter was used because the 59 ends of capped transcripts generated from this promoter with t7 polymerase and the m 7 gpppa cap analog are identical to the 59 end of the genomes of naturally occurring chikv strains. in contrast, capped rnas generated by in vitro transcription from the frequently used sp6 promoter will contain m 7 gpppg at their 59 terminus, i.e. will contain an additional 59terminal g residue. however, existing chikv cdna clones that contain the sp6 promoter also efficiently yield infectious virus and it is assumed that the additional 59-terminal g residue is removed during subsequent rounds of replication. in line with this, in vitro transcribed rna from pchikv-ls2, a variant of plasmid pchikv-ls3 in which the phi2.5 promoter was replaced with the sp6 promoter also yielded infectious virus. plasmid pchikv-ls3-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-ls3, the infectious clone encoding the synthetic 'wild type' strain chikv ls3, and plasmid pchikv-ls3-mcs were generated from pchikv-ls3-gfp by deleting specific restriction fragments, as described in materials and methods (fig. 1) . in the original alignment, strains drde-07 (genbank u372006) and d570/06 (genbank ef012359) shared the highest sequence similarity with ls3, with 3 amino acid differences (table 2) . however, a blast search performed in march 2013, three years after the design of chikv ls3, and alignment of the retrieved complete chikv genomes revealed that strains ind-06-ap3 (genbank ef027134), ind-gj53 (genbank fj000065), and chik31 (genbank eu564335) share the highest degree of nucleotide sequence identity with chikv ls3 (.99.9%), with only 5-7 nucleotide differences respectively (table s1). interestingly, these indian strains were not included in the original alignment on which the ls3 sequence was based, as they do not contain the e1-a226v mutation (table 1) . however, nsp1234 of ls3 is identical to that of ind-06-ap3. at the amino acid level, chikv ls3 differs at 4 positions from lr2006_opy1 and at 3 positions from ita07-ra1 (table 2 ). to determine whether infectious virus could be generated from the synthetic chikv clones, in vitro transcribed rna was electroporated into bhk-21 cells. strong egfp fluorescence was readily detected 16 h after transfection of chikv ls3-gfp rna. for chikv ls3 and ls3-gfp rna specific infectivities of approximately 10 5 pfu/mg of rna were found in infectious center assays, which is similar to what has been found for other chikv cdna clones [17, 18] . virus titers in cell culture supernatants 16 h after electroporation, were generally in the range of 10 5 -10 6 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-21 cells with uncapped chikv rna did not result in the release of infectious virus. to assess the stability of egfp reporter expression, chikv ls3-gfp was serially passaged (moi 0.5) in both 293/ace2 and vero e6 cells. virus harvested during each passage was used to infect vero e6 cells at an moi of 0.2 and immunofluorescence microscopy revealed that at passage 10, over 95% of the e2positive foci still displayed robust egfp expression. sequencing of cdna obtained by rt-pcr amplification of rna extracted from extracellular virions revealed that, after 3 passages on vero e6 cells, the consensus genome sequence of chikv ls3-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 ls3-gfp was able to productively infect hela, mrc-5, huh7, 293, and 293/ace2 cells (data not shown). infection of hela and huh7 cells was not very efficient and these cells were therefore not used for any further experiments. 293/ace2 cells were selected for this study, as they supported high levels of chikv ls3-gfp replication, could be efficiently transfected with sirnas, and -unlike regular 293 cellsadhered well to tissue culture plastics. 293/ace2 cells stably express angiotensin-converting enzyme 2 (ace2), the receptor for sars-coronavirus. ace2 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 ( [34] ; 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 e6, 293/ace2, and c6/36 cells were compared to those of ita07-ra1, which was isolated during the 2007 chikv outbreak in italy ( fig. 2a-c) . the growth curves of chikv ls3 on all three cell lines were found not to differ significantly from those of ita07-ra1, with virus titers reaching a maximum 14-18 h post infection (p.i.). peak virus titers on mosquito cells were approximately 1-log higher than those on mammalian cells. chikv ls3-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 6 h p.i. and peaked around 22 h p.i. the plaque morphology of the synthetic viruses was similar to that of ita07-ra1 (fig. 2d) . chikv ls3 induced a cytopathic effect (cpe) indistinguishable from the natural isolate. on vero e6 cells early signs of cpe started to appear around 12 h p.i. and cpe was complete by 24 h p.i. (fig. 2e) . to study chikv-induced transcriptional host shut-off, the incorporation of 3 h-uridine into cellular and viral rna was analyzed by metabolic labeling of infected 293/ace2 cells at various time points post infection (moi of 5). a strong reduction in the incorporation of 3 h-uridine into rna was observed at 10-12 h p.i. in cells infected with chikv ls3 or ita07, as determined by liquid scintillation counting of total rna samples table 2 . comparison of the amino acid sequences of chikv ls3 and various natural isolates. the chikv ls3 amino acid sequences were aligned with those of several highly similar natural isolates, and clinical isolate nl10/152. only amino acid differences are indicated and identity is represented by dots. the numbering is based on the sequence of ls3, which is also equal to that of lr2006_opy1. it is important to note that -like all other chikv strains in this ( fig. 3a) . inhibition of cellular transcription with actinomycin d for 30 min. prior to metabolic labeling at 12 h p.i. revealed the contribution of viral rna synthesis to the total signal. fluorographic detection of 3 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 6 h p.i (fig. 3b) . transcriptional shut-off occurred around 10-12 h p.i. and was induced by chikv ls3 and ita07 with similar kinetics, although ls3 seemed to act slightly faster. to examine chikv-induced translational shut-off, the synthesis of 35 s-labeled viral and cellular proteins during the course of chikv ls3 infection was analyzed by metabolic labeling of infected 293/ace2 cells with 35 s-met and 35 s-cys (fig. 3c) . a clear shut-off of host translation was observed 8-9 h p.i. beyond 9 h p.i. the bulk of newly produced protein appears to be of viral origin, likely c, e1, e2 and their precursors (indicated with * in fig. 3c ). chikv ita07 and ls3 induced translational host shut-off in a similar manner (only results obtained with ls3 are shown in fig. 3c ). both chikv ita07-ra1 and the synthetic viruses established non-cytopathic persistent infections in c6/36 mosquito cells. all characterization experiments have been performed in both 293/ ace2 and vero e6 cells, with similar results. for simplicity only the results for 293/ace2 cells are shown, except for immunofluorescence experiments, which were done with vero e6 cells as they had a more suitable morphology. the replication cycle of the synthetic viruses and ita07-ra1 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 293/ ace2 cells infected with chikv ls3, ls3-gfp, or ita07-ra1 at various time points post infection. negative-and positivestranded rnas were detected by hybridization with 32 p-labeled oligonucleotide probes (fig. 4a ). both negative-and positivestrand rnas were readily detected in cells infected with the various strains starting at 6 h p.i. the negative-strand rna was less abundant than the positive strand, it was easily detected relatively early in infection ( fig. 4a top panel, fig. 4b) , 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 6 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 6 and 14 h p.i. were treated with singlestrand-specific rnase a/t1 before hybridization, the negativestrand levels at the late time point were approximately 70% of that at 6 h p.i, instead of the approximately 50% in untreated samples (data not shown). using a positive-strand-specific probe, both the 49s genomic and 26s sgrna could be detected, and both rnas accumulated until 12 h p.i (fig. 4a middle panel, fig. 4c ). the ratio of genomic to sgrna varied between 1:3.5 and 1:5.5 during the course of infection, similar to the ratios reported for semliki forest virus and sinv [35] . the kinetics of rna synthesis and rna accumulation levels were similar in chikv ls3-and ita07-ra1-infected cells. in cells infected with chikv ls3-gfp, the additional subgenomic rna encoding the egfp reporter gave rise to an extra band above the 26s rna band, and its expression level was calculated to be approximately half of that of the 26s rna. the individual levels of the two sgrnas expressed by chikv ls3-gfp were lower than those of ita07 or ls3, but their combined abundance was comparable to that of the viruses expressing a single sgrna (fig. 4c ). to monitor viral protein expression, 293/ace2 cells were infected with chikv ls3, ls3-gfp, or ita07-ra1 and total protein was isolated at various time points post infection. these samples were analyzed by western blotting with antisera against the nonstructural proteins nsp1 and nsp4, and the structural protein e2. expression of nsp1, e2, and the e3e2 precursor could be detected as early as 6 h p.i. and the proteins accumulated over time, reaching a plateau around 12 h p.i. (fig. 5) . the rdrp nsp4 could not be detected in infected cells using a chikv nsp4specific 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 nsp4 in infected cells, as was also observed for other alphaviruses [36] . in addition, a quantitative proteomics study on chikv-infected cells also suggested that at 10 h p.i. the amount of nsp4 was at least 200-fold lower than that of nsp1 (treffers, tas, de ru, van veelen, snijder and van hemert, manuscript in preparation). indirect immunofluorescence analysis of vero e6 cells infected with chikv ls3, ls3-gfp, or ita07-ra1 at various time points showed that the localization and expression kinetics of e2 and dsrna were similar for the natural isolate and the synthetic viruses (fig. 6) . double-stranded rna, which is assumed to be generated during replication of chikv in infected cells [37] , could be detected as early as 4 h p.i. and remained clearly visible throughout the infection. the dsrna localized to foci throughout the cytoplasm. the e2 protein could be detected from 6 h p.i. onwards with maximum expression reached by 12 h p.i. the e2 protein mainly localized to the plasma membrane of infected cells. egfp produced by the reporter virus was visible from 6 h p.i. onwards, reaching a maximum level around 12 h p.i. (fig. 6c ). chikv ls3 and ita07-ra1 were compared in a neutralization assay using the neutralizing monoclonal antibody 1h7 that was raised in mice against chikv ita07-ra1 virions, and appears to recognize a linear epitope in e2 [31] . the nonneutralizing mab 3h9 was used as a control. both the natural isolate and chikv ls3 were neutralized with similar characteristics by 1h7, while their infectivity was not affected by 3h9 (fig. 7) . newborn mice are highly susceptible to chikv infection and they develop symptoms as lethargy, dragging of hind limbs, flaccid paralysis, and reduced weight gain [38] . 12-day old mice were injected intraperitoneally with 100 tcid 50 of chikv ls3, ls3-gfp or prototype strain s27 as a control. the animals were euthanized when their humane end points were reached 3 or 4 days post infection and viral rna levels in brain tissue were analyzed (fig. 8) . both synthetic viruses behaved like the natural isolate in vivo, causing lethal infections with similar kinetics (fig. 8a ). in addition, the viral titers in the brains of chikv s27-infected mice were similar to those of mice infected with the synthetic viruses (fig. 8b ). to evaluate their suitability for analyzing the potency and mechanism of action of antiviral compounds, the sensitivity of chikv ls3 and ls3-gfp to a number of such compounds was determined and compared to ita07-ra1. 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 32 mm (data not shown). the compounds 3-deaza-adenosine, 6-aza-uridine, chloroquine, and mycophenolic acid were tested in cpe reduction assays with vero e6 cells infected at an moi of 0.005 and analyzed 4 days p.i. they were all found to inhibit chikv replication with ic 50 s in the low micromolar range and with minimal cytotoxicity (fig. 9a-d) . no substantial differences were observed between the ic 50 values calculated for ita07-ra1, ls3 and ls3-gfp. the four compounds also clearly reduced egfp reporter gene expression in vero e6 cells infected with chikv ls3-gfp (fig. 9f) . slightly lower ic 50 values were obtained for 6-aza-uridine and chloroquine, and a significantly higher ic 50 was observed for 3-deazaadenosine in this assay, compared to the cpe-based assay. this might be due to the mode of action of 3-deaza-adenosine and/or due to differences in experimental set-up compared to the cpebased assay (moi 0.05 vs. 0.005; measurement 42 h p.i. vs. 4 d p.i). ribavirin is a known inhibitor of chikv replication, but in our cpe reduction assay with vero e6 cells it was not very effective in inhibiting replication of the various strains, as ic 50 values of over 400 mm were obtained (fig. 9e, gray lines) . this is likely due to the inefficient conversion of ribavirin to its active phosphorylated form in vero e6 cells [39] . therefore, we have also analyzed the effect of ribavirin in a 2-day cpe reduction assays with bhk-21 cells, which are able to metabolize ribavirin [40, 41] and found ic 50 s of 15-21 mm for the various strains. clinical isolate nl10/152 was also included in the assays and appeared to be somewhat more sensitive to the antiviral compounds than ls3 and ita07-ra1. however, the slower replication kinetics of this strain made it impossible to directly compare nl10/152 and ls3 in the same cpe reduction assays, despite the fact that virus yields and cytopathic effect of nl10/152 and ls3 were comparable (data not shown). the massive chikv outbreaks that have been occurring in asia and the indian ocean region since 2005 are associated with the emergence of strains with the a226v substitution in the e1 glycoprotein, which allowed their transmission by a novel mosquito vector, aedes albopictus [8] [9] [10] [11] [12] [13] . 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 ls3 sgrna at 12 h p.i (100%). genomic rna levels are indicated with black lines, sgrna levels with gray lines. the total level of both sgrnas expressed by ls3-gfp is indicated with the gray dotted line. doi:10.1371/journal.pone.0071047.g004 asian lineage chikv strains that have been endemic in the region for decades. since the 1980s, 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 2007 and france in 2010 [15, 16] and recent studies suggest that also the usa is at risk for locally transmitted chikv outbreaks [42, 43] . 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 [17] [18] [19] [20] [21] [22] [23] . 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 [24] . 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 [44] . 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 [44] . 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 e1-226v chikv strains that were circulating during the 2005-2009 outbreaks. therefore, we have constructed a fully synthetic cdna clone, chikv ls3, based on the consensus sequence of the aligned genomes of these recent e1-226v 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 ls3-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 148 complete chikv genomes that were in genbank by june 2013 yielded a consensus sequence that differed only at 3 nucleotide positions from the sequence of ls3 that was designed 3 years earlier. these were position 7,435 at which we introduced a translationally silent restriction site (g7435a), a synonymous urc substitution at position 3,397, and position 10,670, which is a c in 68% of all deposited genomes (strains with e1-226a), while the remaining (e1-226v) strains have a u at this position. an interesting observation was that 6% of the sequenced chikv strains, including the prototype strains s27 and ross, contain an arginine codon instead of the opal stop codon that is present between the nsp3-and nsp4-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 [45] . this might also explain why the sequence of the original clinical isolate of lr2006-opy1 (genbank dq443544.2) contains the opal termination codon near the end of the nsp3 coding region, while the infectious clone of this strain (genbank eu224268.1) 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 ita07-ra1. like the natural isolate, the synthetic viruses caused cytopathic infections in vero e6 and 293/ace2 cells (fig. 2e) , whereas non-cytopathic persistent infections were observed in the mosquito cell line c6/36. in vertebrate cells all strains caused a shut-off of cellular translation around 8-9 h p.i. and a strong inhibition of cellular transcription by 10-12 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 ita07-ra1 ( fig. 2-6 ). in addition, the synthetic viruses caused lethal infections in 12-day old mice, with virus spreading to the brain, as observed for natural isolates (fig. 8) . 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 ls3-gfp was assessed and after 3 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 10 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 ls3 and ls3-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 ita07-ra1. the lysosomotropic agent chloroquine and nucleoside analog 6-aza-uridine inhibited the replication of the synthetic viruses and natural isolates with ic 50 s that were in the same range and comparable to values previously reported by others [46] [47] [48] [49] [50] [51] . 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 [46] . the nucleoside analog 6-aza-uridine has previously been reported to inhibit the replication of a variety of viruses, including chikv [47, 51] . 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 [47, 52] . 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 [39] . 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 [40, 41, 53] . in our hands, ribavirin inhibited chikv replication in bhk-21 cells with an ic 50 of around 18 mm, while it was hardly effective in vero e6 cells, with ic 50 values of over 400 m. the ic 50 that we obtained with bhk-21 cells is in the same range as those previously reported for the antiviral effect of ribavirin on chikv replication [47, 51] . cyclosporin a, which through its effect on the cellular cyclophilins, inhibits the replication of a variety of viruses (for recent review see [54] ), had no effect on chikv replication. we identified 3-deaza-adenosine as a novel inhibitor of chikv replication with an ic 50 of approximately 6 mm and a cc 50 .50 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 [55] ). the antiviral activity of 3-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 [55] . 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 3-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 50 values calculated for ita07-ra1, ls3 and ls3-gfp, indicating that the synthetic viruses are suitable for use in antiviral screens. for most compounds, a faster and simpler assay with chikv ls3-gfp reporter virus showed a good dosedependent response that correlated well with results obtained in the cpe-based assay. clinical isolate nl10/152 exhibited slightly slower replication kinetics and appeared to be more sensitive to antiviral compounds than ita07 and the synthetic viruses. differences in the sensitivity to antiviral compounds among clinical isolates is not an uncommon phenomenon. nl10/152 differs at 7 amino acid positions from ls3 and it will be interesting to determine the contribution of these mutations, in particular the r88s substitution in nsp4, 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 s1 comparison of chikv ls3 with the genome sequences of various closely related natural isolates. only differences between ls3 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 ls3. genomes were aligned with mafft and analyzed in jalview. numbering is based on the sequence of lr2006_opy1 (and is equal to ls3 numbering). the nucleotide at position 10670 (indicated in gray) determines whether the strain has the a226v mutation in the e1 protein. strains with a t at this position have the a226v mutation. differences not included in the comparison are the 35 nt, 5 nt and 23 nucleotides that are missing from the 3'utr of the sequences of drde-07, d570/06 and ita07-ra1, respectively. the missing first 19 nt, missing last 13 nt and the insertion of an a after position 11564 in the sequence of ind-06-ap3 were also not included in this comparison. 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virus in the united states host alternation of chikungunya virus increases fitness while restricting population diversity and adaptability to novel selective pressures genetic and fitness changes accompanying adaptation of an arbovirus to vertebrate and invertebrate cells on chikungunya acute infection and chloroquine treatment inhibitors of alphavirus entry and replication identified with a stable chikungunya replicon cell line and virus-based assays characterization of reemerging chikungunya virus chikungunya disease and chloroquine treatment assessment of in vitro prophylactic and therapeutic efficacy of chloroquine against chikungunya virus in vero cells in vitro inhibition of chikungunya and semliki forest viruses replication by antiviral compounds: synergistic effect of interferon-alpha and ribavirin combination cellular impdh enzyme activity is a potential target for the inhibition of chikungunya virus replication and virus induced apoptosis in cultured mammalian cells cell type mediated resistance 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 e2 antiserum and to adriaan de wilde and emmely treffers for helpful discussions and sharing their unpublished data. key: cord-316908-8ti75mru 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: 2020-02-10 journal: vet res doi: 10.1186/s13567-020-0739-7 sha: doc_id: 316908 cord_uid: 8ti75mru 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-j2 cells. we first characterized the kinetics of pedv entry into cells and found that the highest invasion rate of pedv was approximately 33% in the ipec-j2 cells and approximately 100% in the vero cells. to clarify the specific endocytic pathways, systematic research methods were used and showed that pedv enters cells via the clathrinand caveolae-mediated endocytosis pathways, in which dynamin ii, clathrin heavy chain, eps15, cholesterol, and caveolin-1 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 2010. pedv was first reported in 1971 in the uk [1] , and afterward was also discovered in europe and asia [2] [3] [4] [5] . although pedv has persisted in asian swine-producing countries, it does not attract enough global attention. after october 2010, severe ped outbreaks occurred even in chinese pig farms that were already vaccinated with cv777inactivated or live-attenuated vaccines [2, [6] [7] [8] . in 2013, the first pedv outbreak occurred in the us and rapidly spread across the entire country [9] . molecular epidemiological results show the genetic differences between classical (gi subtype) and new pedv variant strains (gii subtype) [2, 3, 5, 6, 8, 9] . pedv is presently recognized worldwide due to dramatic changes observed in its epidemic character, pathogenic properties, and gene drift [10] . studies focused on its pathogenesis [11, 12] , immune evasion [13] and developing effective vaccines [14, 15] 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 [16, 17] . 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 [18, 19] . caveolae is a plasma-specific invagination structure with a diameter of 50-100 nm. when viral particles interact with receptors, caveolae coated with caveolin-1 invaginates and pinches off plasma membrane, then the caveolae vesicles mature into caveosomes and deliver "cargoes" to early endosomes [20, 21] . 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 [22] . 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 hepg2 and cos7 cells is clathrindependent while entry into vero e6 cells is clathrin-and caveolae-independent [23, 24] , but the lipid raft plays an important role in the process [25] . mhv entry into cells needs clathrin [26] [27] [28] , the same as hcov-nl63 [29] . for hcov-229e, caveolae-mediated endocytosis is utilized to enter human fibroblast cells [30] . 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 [31] . 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 [32] . 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 2014, park et al. [33] 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-j2 cells as models for pedv entry and chose cv777-like strain gds09 and highly pathogenic variant strain gds01 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 10% fetal bovine serum (fbs, gibco) and antibiotics (100 u/ml penicillin and 100 μg/ml streptomycin). ipec-j2 cells were grown in dmem/nutrient mixture f-12 (dmem/f12) supplemented with 5% fbs and antibiotics. the pedv strains used in this study were cv777-like strain gds09 (gi subtype, genbank id: mh726408.1) and highly pathogenic variant strain gds01 (gii subtype, genbank id: km089829.1). exogenous trypsin (10 μg/ml) was added to proliferate pedv strains in vero cells and 5 μg/ ml trypsin was added in ipec-j2 cells. sbti (soy bean trypsin inhibitor type i; sigma no. t6522). the endocytic inhibitors used included dynasore (sigma-aldrich, no. 324410), chlorpromazine (cpz, sigma-aldrich, no. c0982), methyl-β-cyclodextrin (mβcd, sigma-aldrich, no. c4555), nystatin (sigma-aldrich, no. 475914), ammonium chloride (nh4cl, sigma-aldrich, no. a9434), and bafilomycin a1 (baf a1, sigma-aldrich, no. 196000). antibodies against clathrin heavy chain, caveolin 1, eea1, rab7, and lamp1 coupled with secondary goat anti-rabbit alexa fluor 488 and goat anti-mouse alexa fluor 594 were purchased from abcam. the mouse anti-pedv-s monoclonal antibody [34] 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 15 (gfp-eps15-wt and gfp-eps15-m), and caveolin-1 (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 6-well plates until confluence. after washed with pbs, cells were infected with pedv strains at a multiplicity of infection (moi) of 0.5 with or without trypsin (10 μg/ml) or with trypsin and 25 μg/ml sbti for 12 h before the quantification of the viruses by qrt-pcr. to test the dynamics of pedv internalization, vero or ipec-j2 cells were seeded in 12-well plates until confluence. the cells were pre-chilled for 10 min and inoculated with pedv at a moi of 0.5 at 4 °c for 1 h for virus binding. the cells were washed three times with ice-cold pbs to remove unbounded viruses and immediately warmed to 37 °c to initiate internalization. after incubation for the indicated time intervals, the cells were treated with proteinase k (1 mg/ml) at 4 °c for 30 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 [34] . to test the effect of inhibitors on pedv internalization, it was necessary to evaluate the cytotoxicity of cell inhibitors. the cells were seeded in 96-well plates at a density of 2 × 10 5 cell/well, grown for 24 h, and treated with endocytic inhibitors at the indicated concentration for 4 h. then 10 μl of cck-8 solution was added to each well and incubated at 37 °c for 1 h. an absorbance of 450 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 1 h and then infected with gds01 or gds09 strains at moi = 1 in the presence of drugs for 1 h. after washing with citrate buffer (ph 3.0) [35] and pbs, the cells were incubated with medium containing trypsin for 6 h or 9 h at 37 °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 480 real-time pcr system (roche diagnostics, indianapolis, in, usa). for the western blotting analysis [36] , the cells were washed with pbs and lysed in ripa lysis buffer on ice for 30 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-j2 cells with the overexpression plasmids of wild-type and mutant dynamin ii (gfp-dyn-wt and gfp-dyn-m), eps 15 (gfp-eps15-wt and gfp-eps15-m), and caveolin-1 (gfp-cav-wt and gfp-cav-m) were performed using lipofectamine 2000 (invitrogen) transfection reagents according to the manufacturer's protocol. the cells were seeded in 12-well plates until 80% confluence. 24 h after transfection, the cells were infected with pedv at moi = 1 for 1 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: 5′-cac ctc atg atc aat aac a-3′, chlorocebus sabaeus 5′-cct aca tca aca cga acc a-3′), clathrin heavy chain (sichc, sus scrofa 5′-ccc ata cca tga ctg atg a-3′, chlorocebus sabaeus 5′-gat gaa cct tat gca tgc a-3′), eps 15 (sieps15, sus scrofa 5′-cct gtg gat att ctt gga a-3′, chlorocebus sabaeus 5′-ccc aga aac agc aag tac a-3′), and caveolin-1 (sicav, sus scrofa 5′-caa cat gca gaa aga aat a-3′, chlorocebus sabaeus 5′-cct tca ctg tga cga agt a-3′) were designed and synthesized based on the corresponding full-length mrna sequences of sus scrofa and chlorocebus sabaeus, sir-nas against rab7a (sirab7, 5′-gat ggt gga tga cag act a-3′), and vps39 (sivps39, 5′-gct tca aga gag act act a-3′) 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 12-well plates until 80% confluence. to ensure transfection efficiency, a second transfection was carried out at 24 h after the first transfection. at 48 h post-first transfection, the cells were infected with pedv at moi = 1 for 1 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 6 hpi and 9 hpi, respectively. alexa-594 labeled transferrin (trf ) or alexa-555 labeled cholera toxin b subunit (ctb) were diluted at 1:500 and mixed with pedv at moi = 10. the cells were washed three times with pbs and added to the mixture of pedv and trf or ctb at 4 °c for 1 h and then incubated at 37 °c for 30 min for internalization. after washing with pbs, the cells were fixed, permeabilized, blocked, incubated with mouse anti-pedv-s monoclonal antibody, incubated with alexa 488-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 12 h were washed with ice-cold pbs and incubated with pedv at 4 °c for 1 h. cold viruses were replaced with pre-warmed medium, and the cells were immediately shifted to 37 °c. at specific time points, the cells were fixed in 4% paraformaldehyde at rt for 15 min after washing three times with pbs. permeabilization was carried with 0.5% triton x-100 at rt for 15 min. after washing with pbs, the cells were blocked with 5% bsa in pbst at rt for 60 min to block unspecific binding sites. the specific primary antibodies against chc, eea1, caveolin-1, rab7, lamp1, and anti-pedv-s antibody were used to probe the cells at 4 °c overnight. the cells were incubated with secondary antibodies (goat anti-rabbit igg antibody conjugated to alexa fluor 488 and goat anti-mouse igg antibody conjugated to alexa fluor 594) at 37 °c for 1 h. fluorescent images were acquired using the light-scanning module of a leica tcs sp8 sted 3× confocal microscope. the cells (5 × 10 7 ) were incubated or not incubated with pedv at 37 °c for 1 h, washed three times with ice-cold pbs, and lysed in 1 ml tne buffer (25 mm tris, 150 mm nacl, 5 mm edta, and ph 7.5) containing 1% triton x-100 and 1% phenylmethanesulfonyl fluoride (pmsf) on ice for 30 min. the homogenized cell lysates were centrifuged at 4 °c for 5 min at 1000 g and the supernatant was mixed with isometric 1 ml containing 80% sucrose in tne buffer. the lysates-sucrose mixture was placed at the bottom of ultracentrifugal tubes and overlaid with 7 ml 30% and 3 ml 5% sucrose in tne buffer. the cell lysates were ultracentrifuged at 4 °c for 16 h at 20 000 g in a sw41 rotor (beckman). after centrifugation, twelve 1 ml fractions were collected from the top to the bottom of the tubes. the fractions were concentrated with 6% peg at 4 °c overnight, and the pellets were resuspended in 100 μl of tne buffer after centrifuging at 4 °c for 30 min at 10 000 g. the localizations of lipid raft-associated protein caveolin-1 and pedv n protein were analyzed by western blotting. all the graphs were created with graphpad prism 6 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 0.05 were defined as the threshold for statistical significance. p values between 0.05 and 0.01 were marked with one asterisk, p values between 0.01 and 0.001 were marked with two asterisks, p values between 0.001 and 0.0001 were marked with three asterisks, and p values less than 0.0001 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 1a , gds01 strain needed trypsin while gds09 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 = 0.5 at 4 °c for adsorption and shifted to 37 °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 1 ) showed that most of the pedv particles were detached from the cells by proteinase k at the beginning of invasion. after 60 min in the vero cells ( figure 1b ) and 45 min in the ipec-j2 cells ( figure 1c ), nearly maximum proportions of viral particles completed the internalization. approximately 95% of the pedv particles entered the vero cells, while only 30% entered the ipec-j2 cells. notably, the gds01 strain demonstrated less efficient invasion than the gds09 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 [35, 37] . 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 [38] , 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 50 μm of dynasore had no effect on the viability of the vero and ipec-j2 cells (additional file 1). cells were pre-treated with 30 μm and 50 μm dynasore for 1 h before pedv infection. dmso was used as a negative control. the effect of dynasore on pedv entry was quantified by qrt-pcr at 6 hpi. pedv invasion was significantly inhibited by dynasore. at a concentration of 50 μm, the invasion rates of the gds01 and gds09 strains into the vero cells were approximately 17% and 12%, and the invasion rates into the ipec-j2 cells were 8% and 63%, respectively ( figure 2a) . a comparison of the invasion rates of the gds01 and gds09 strains indicated that the gds01 strain was more sensitive to dynasore than the gds09 strain when invading the ipec-j2 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 [39, 40] . mutation of dynamin ii from 44k to 44a can inhibit gtpase activity and reduce endocytosis [41] . cells were transfected with wild-type and mutant types of dynamin ii respectively and infected with gds01 and gds09 strains at 24 h after transfection. the confocal results showed that the vero and ipec-j2 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 2b ). sirna interference was also used to identify the importance of dynamin ii on virus entry [23, 35, 42] . 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-j2 cells was obvious at both the mrna and protein levels (additional file 1). cells were infected with pedv after transfection twice and the internalized virions were quantified at 6 hpi and 9 hpi by qrt-pcr and western blotting assay, respectively. the qrt-pcr results ( figure 2c ) showed that the knockdown of dynamin ii expression reduced the pedv internalization. the internalization rates of the gds01 and gds09 strains into vero cells were approximately 50% and 57%, the internalization rates into the ipec-j2 cells were approximately 48% and 60%, respectively, but there was no significant difference between the gds01 and gds09 strains in the two cells ( figure 2c ). the same results were confirmed by western blotting ( figure 2d ). 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-j2 cells with pedv and trf, which is the most typical biomolecule that uses cme to enter cells [43] . the co-inoculation results figure 1 trypsin-dependency and kinetics of pedv entry into cells. a vero cells were seeded in 6-well plates until confluence. cells were washed with pbs and infected with pedv strains (moi = 0.5) without trypsin or in the presence of trypsin (10 μg/ml) or trypsin and 25 μg/ml sbti. cells were collected for qrt-pcr at 12 hpi. b, c vero cells (b) and ipec-j2 cells (c) were incubated with pedv gds01 and gds09 strains, respectively, at 4 °c for 1 h and shifted to 37 °c immediately to initiate internalization. at 0, 15, 30, 45, 60, 75, 90, 105, and 120 min after incubation, the cells were treated with proteinase k (1 mg/ml) at 4 °c for 30 min to inactivate the non-internalized virions. the control cells were washed with pbs. the invasion rates were calculated by qrt-pcr analysis. ****p < 0.001. dynamin ii involved in pedv entry. a cells were pre-treated with 30 μm and 50 μm dynasore at 37 °c for 1 h, respectively, and incubated with gds01 or gds09 strain for 1 h. dmso was used as a negative control. the cells were collected at 6 hpi for qrt-pcr assay to test the invasion efficiency of pedv. b vero and ipec-j2 cells were transfected with gfp-dyn-wt and gfp-dyn-m, respectively, and infected with pedv strains at 24 h after transfection. the cells were fixed at 12 hpi and stained for confocal analysis. c, d vero and ipec-j2 cells were transfected with sidyn twice and infected with pedv strains at 24 h after the second transfection. the invasion rates of pedv into the cells were detected at 6 hpi and 9 hpi for qrt-pcr and western blotting analysis, respectively. ctrl means control. scale bars indicate 25 μm. **0.05 < p < 0.01; ***0.01 < p < 0.001; ****p < 0.001. showed that pedv co-located with trf in the two types of cells ( figure 3a ), 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 15, the knockdown expression of chc and eps 15 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 [44] . the cytotoxicity test showed that 30 μm cpz have no effect on the viability of the vero cells and 50 μm cpz have no effect on the viability of the ipec-j2 cells (additional file 2). vero and ipec-j2 cells were treated with cpz at different concentrations for 1 h and then infected with pedv. the internalization rates of pedv after cpz treatment were quantified by qrt-pcr and western blotting at 6 hpi and 9 hpi, respectively. the qrt-pcr results (figure 3b ) showed that pedv invasion was significantly inhibited by cpz. the invasion rates of the gds01 and gds09 strains at the highest drug concentrations were nearly 48% and 23% in the vero cells and 24% and 50% in the ipec-j2 cells, respectively. notably, there were no significant differences between the gds01 and gds09 strains in the vero cells but the gds01 strain was more sensitive than the gds09 strain in the ipec-j2 cells, reflecting the gds01 strain's significantly decreased invasion rates ( figure 3b ). the same results were also observed by western blotting ( figure 3c ) and ifa assay (additional files 3, 4) . the role of cme in endocytosis was also identified by the overexpression of gfp-tagged dominant negative mutants of eps 15 [45] . eps 15 is a critical component of ccps by interacting with adaptor protein 2 (ap-2), a major clathrin adaptor complex [46] . cells transfected with wild-type (gfp-eps15-wt) and mutant eps 15 (gfp-eps15-m) were infected with pedv strains at 24 h after transfection. the confocal results of the pedv invasion showed that the cells overexpressing wild-type eps 15 were infected with pedv while few infections were observed in the overexpressed gfp-eps15-m cells ( figure 3d ). 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 15. chc and clathrin light chain form a triskelion shape, which is a key component for regulating the formation and disassembly of the clathrin lattice [47] . 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 6 hpi and 9 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 gds01 and gds09 strains were 50% and 62% in the vero cells and 61% and 65% in the ipec-j2 cells, respectively, and there was no significant difference between the gds01 and gds09 strains ( figure 3e ). knockdown of the expression of eps 15 also significantly reduced the invasion rates of pedv. the invasion rates of the gds01 and gds09 strains were 61% and 65% in the vero cells and 51% and 66% in the ipec-j2 cells, respectively, and there was no significant difference between the gds01 and gds09 strains ( figure 3g ). the significant inhibition of sichc and sieps15 on pedv entry was also observed by western blotting assay (figures 3f, h) . to estimate whether pedv directly entered the cells through cme, we analyzed the localization of pedv and chc in the vero and ipec-j2 cells, respectively. pre-cooled cells were incubated with pedv at 4 °c for 1 h for adsorption and shifted to 37 °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-j2 cells ( figure 3i ), 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 [48] . many studies showed that most enveloped virus relied on cholesterol to invade cells [49, 50] . 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 [51] . nystatin can bind to the cholesterol-enriched regions of cell membrane and then decompose cholesterol and impair cholesterol synthesis [52] . the cytotoxicity test showed that the maximum tolerance concentrations of vero and ipec-j2 cells to mβcd were 3 mm and 1.5 mm, respectively, and the maximum tolerance concentrations to nystatin were 30 μm and 50 μm, respectively (additional file 5). cells were pre-treated with different concentrations of mβcd and nystatin for 1 h, then infected with pedv. the effects of drugs on pedv entry were estimated by qrt-pcr and western blotting at 6 hpi and 9 hpi, respectively. mβcd showed a significant inhibition of pedv entry. the internalization rates of the gds01 and gds09 strains after mβcd treatment were approximately 4% and 6% in the vero cells and approximately 5% and 35% in the ipec-j2 cells. there were no significant differences between the gds01 and gds09 strains in the mβcd-treated vero figure 3 pedv entry relies on the cme pathway. a vero cells and ipec-j2 cells were incubated with mixture of alexa-594 labeled trf (red) and pedv (green) at 4 °c for 1 h, and then shifted to 37 °c for 30 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 10 μm and 30 μm of cpz, and the ipec-j2 cells were pre-treated with 30 μm and 50 μm of cpz, respectively, at 37 °c for 1 h and incubated with gds01 or gds09 strains for 1 h. double-distilled water was used as a negative control. the cells were collected at 6 hpi and 9 hpi for qrt-pcr and western blotting assay, respectively, to test the invasion efficiency of pedv. d the vero cells (left) and ipec-j2 cells (right) were transfected with gfp-eps15-wt and gfp-eps15-m, respectively, and infected with pedv strains at 24 h after transfection. the cells were fixed at 12 hpi and stained for confocal analysis. e-h the vero cells and ipec-j2 cells were transfected with sichc and sieps 15 and infected with pedv strains at 24 h after the second transfection. the cells were collected at 6 hpi and 9 hpi for qrt-pcr and western blotting analysis, respectively. i the cells were pre-cooled at 4 °c for 15 min, incubated with pedv strains at 4 °c for 1 h, shifted to 37 °c for 5 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 50 μm in a, 25 μm in d, and 5 μm in i. **0.05 < p < 0.01; ***0.01 < p < 0.001; ****p < 0.001. cells but in the ipec-j2 cells, the gds01 strain was more sensitive to mβcd ( figure 4a ). the results were confirmed by western blotting ( figure 4b ) and ifa assay (additional files 3 and 4). pedv entry was significantly inhibited by nystatin treatment. the internalization rates of the gds01 and gds09 strains after nystatin treatment were approximately 75% and 25% in the vero cells and approximately 65% and 45% in the ipec-j2 cells. the gds09 strain was more sensitive to nystatin than the gds01 strain in both the vero and ipec-j2 cells ( figure 4c ). the same results were confirmed by western blotting ( figure 4d ) and ifa assay (additional files 3 and 4). 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 6 hpi. the results showed that adding exogenous cholesterol could significantly increase the invasion rate of pedv. the average invasion rate of the gds01 and gds09 strains increased from 10% to over 90% in the vero cells and from 10% and 50% to approximately 95% in the ipec-j2 cells ( figure 4e ). 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-j2 cells. as both caveolae and lipid raft are rich in cholesterol, they are sensitive to cholesterol inhibitors [53, 54] . 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 [55] . the localization results showed that pedv co-localized with ctb in vero and ipec-j2 cells ( figure 5a ), which means pedv may utilize caveolae-mediated endocytosis to enter cells. as the caveolae is mainly coated with caveolin-1 [56] , knocking down the expression and separating the interaction factors with caveolin-1 blocked the caveolae-mediated endocytic pathway. overexpression of the domain-negative mutant of caveolin-1 [57] blocked its interaction with the interaction factors. cells were transfected with wild-type caveolin-1 (gfp-cav-wt) and mutant caveolin-1 (gfp-cav-m), then infected with pedv at 24 h after transfection, and fixed for confocal observation at 12 hpi. the results showed that pedv infected gfp-cav-wt-overexpressing cells but barely infected gfp-cav-m-overexpressing vero or ipec-j2 cells ( figure 5b ). sirnas (sicav) were designed and synthetized to knockdown caveolin-1 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 6 hpi and 9 hpi, respectively. the qrt-pcr results showed that the knockdown of caveolin-1 expression reduced the internalization of pedv. the inhibition rates in the gds01 and gds09 strains were 53% and 32% in the vero cells and 33% and 40% in the ipec-j2 cells, respectively (figure 5c) . compared with the gds09 strain, the gds01 strain showed a higher degree of reduction in the invasion rate in the vero cells but there was no significant difference in the ipec-j2 cells ( figure 5c ). the same results were confirmed by western blotting assay ( figure 5d ). to identify the role of caveolae in pedv entry, we investigated the cellular localization of pedv with caveolin-1. pre-cooled cells were incubated with pedv at 4 °c and then shifted to 37 °c for internalization. the cells were then washed and fixed for 10 min for observation with an ultrahigh-resolution laser confocal microscope. the cellular localization results showed that pedv was colocated with caveolin-1 in the vero and ipec-j2 cells ( figure 5e ). 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 [58] . 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 12 fractions were obtained for western blotting analysis. caveolin-1 was used as the protein marker representing the lipid raft layer [58] . 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 6a ) and virions were detected in both the upper and lower components in the ipec-j2 cells ( figure 6b ). the differences between the gds01 and gds09 strains were the proportion of virions in the upper and lower components in the ipec-j2 cells. the gds01 particles were mainly present in the upper layer while amounts of gds09 particles were present in the lower layer (figure 6b) . 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 [59] . 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 4 pedv entry relies on cholesterol. a, b vero cells and ipec-j2 cells were pre-treated with 1.5 mm and 3 mm and 1 mm and 1.5 mm mβcd, respectively, at 37 °c for 1 h and incubated with gds01 or gds09 strains for 1 h. double-distilled water was used as a negative control. the cells were collected at 6 hpi and 9 hpi for qrt-pcr and western blotting assay, respectively, to test the invasion efficiency of pedv. c, d the vero cells and ipec-j2 cells were pre-treated with 10 μm and 30 μm and 30 μm and 50 μm of nystatin, respectively, at 37 °c for 1 h and incubated with gds01 or gds09 strains for 1 h. dmso was used as a negative control. the cells were collected at 6 hpi and 9 hpi for qrt-pcr and western blotting assay, respectively, to test the invasion efficiency of pedv. e the vero cells and ipec-j2 cells were pre-treated with different concentrations of mβcd at 37 °c for 1 h, supplemented with 400 μg/ml of soluble cholesterol at 37 °c for 1 h, and infected with pedv strains for 1 h. the cells were collected at 6 hpi for qrt-pcr assay to test the invasion efficiency of pedv. *p < 0.05; **0.05 < p < 0.01; ***0.01 < p < 0.001; ****p < 0.001. a vero cells and ipec-j2 cells were incubated with a mixture of alexa-555 labeled ctb (red) and pedv (green) at 4 °c for 1 h, and then shifted to 37 °c for 30 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-j2 cells (down) were transfected with wild-type caveolin-1 (gfp-cav-wt) and domain negative mutant of caveolin-1 (gfp-cav-m), respectively, and infected them with pedv strains at 24 h after transfection. the cells were fixed at 12 hpi and stained for confocal analysis. c, d the vero cells and ipec-j2 cells were transfected with sicav twice and infected with pedv strains at 24 h after the second transfection. the cells were collected at 6 hpi and 9 hpi for qrt-pcr and western blotting analysis, respectively. ctrl means control. e cells were pre-cooled at 4 °c for 15 min, incubated with pedv strains at 4 °c for 1 h, shifted to 37 °c to initiate internalization for 10 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-1 (green) primary antibodies. scale bars indicate 50 μm in a, 25 μm in b, and 5 μm in e. *p < 0.05; **0.05 < p < 0.01; ***0.01 < p < 0.001; ****p < 0.001. 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. nh4cl is an inhibitor of endosome acidification [60] . baf a1 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 [61] . cells were pre-treated with different concentrations of nh4cl and baf a1 (additional file 6) and infected with pedv (additional file 4). the invasion rates of pedv were measured by qrt-pcr and western blotting at 6 hpi and 9 hpi, respectively. the results showed that cells pretreated with nh4cl significantly inhibited pedv entry. the invasion rates in the gds01 and gds09 strains were approximately 30% and 15% in the vero cells and 50% and 73% in the ipec-j2 cells ( figure 7a ). this significant inhibition was also confirmed by western blotting (figure 7b ) and ifa assay (additional files 3 and 4). baf a1 can significantly inhibit pedv entry. the invasion rates in the gds01 and gds09 strains were approximately 75% and 5% in the vero cells and 41% and 30% in the ipec-j2 cells ( figure 7c ). western blotting ( figure 7d ) and ifa assay (additional files 3, 4) also confirmed the inhibition of baf a1. 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 rab5, incorporate rab7, and prepare to fuse with lysosomes [62] . to explore whether pedv particles are trafficked after internalization, we interfered with the expression of rab7 [63] involved in late endosomes and vps39 [32] 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 rab7 (sirab7) can significantly inhibit the invasion efficiency of pedv. the invasion rates of the gds01 and gds09 strains were 49% and 51% in the vero cells and 82% and 38% in the ipec-j2 cells ( figure 8a) . similarly, the knockdown of the expression of vps39 (sivps39) also significantly inhibited the invasion efficiency of pedv. the invasion rates of the gds01 and gds09 strains were 68% and 56% in the vero cells and 45% and 50% in the ipec-j2 cells ( figure 8b ). for cellular location by pedv observation, pre-cooled cells were incubated with pedv at 4 °c and then shifted to 37 °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 eea1, the early endosome protein marker, in the vero were incubated or not with pedv at 37 °c for 1 h and then lysed in tne buffer containing 1% triton x-100 and 1% pmsf on ice for 30 min. after mixing with isometric 80% sucrose, the homogenized cell lysates were subjected to ultracentrifugation after being overlaid with 30% and 5% sucrose. after centrifugation, a total of 12 fractions were collected from the top to the bottom of the tubes. the localizations of the lipid raft-associated protein caveolin-1 and pedv n protein were analyzed by western blotting after being concentrated with 6% peg6000. and ipec-j2 cells 30 min after endocytosis ( figure 8c ) and could co-locate with rab7, the late endosome protein marker, 40 min after endocytosis ( figure 8d ), while the late endosomes were mostly in the form of mvb. colocalization of pedv with lamp1 (lysosomal associated membrane protein 1), an important lysosome membrane component, was also observed 50 min after endocytosis in the two types of cells ( figure 8e ). 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 2010, pedv has attracted global attention. many studies have reported that vaccines based on cv777 or cv777-like strains have low protection efficiency against re-emerging variant strains [3, 4, [6] [7] [8] . genotyping showed that pedv strains can be sorted into two genotypes, gi subtypes (classical) and gii subtypes (variant). the nucleotide sequence of s1 subunit of s protein is 75-90% similar between gi and gii, which shows high variability [10, 64] . as the main antigen of pedv, the s protein incubated with gds01 or gds09 strains for 1 h. double-distilled water was used as a negative control. the cells were collected 6 hpi and 9 hpi for qrt-pcr and western blotting assay, respectively, to test the invasion efficiency of pedv. c, d the vero cells and ipec-j2 cells were pre-treated with 200 nm and 400 nm baf a1 at 37 °c for 1 h and then incubated with gds01 or gds09 strains for 1 h. dmso was used as a negative control. the cells were collected 6 hpi and 9 hpi for qrt-pcr and western blotting assay, respectively, to test the invasion efficiency of pedv. **0.05 < p < 0.01; ***0.01 < p < 0.001; ****p < 0.001. vero cells and ipec-j2 cells were transfected with sirab7 and sivps39 twice, respectively, and then infected with pedv strains at 24 h after the second transfection. the cells were collected at 6 hpi for qrt-pcr analysis. ctrl means control. c-e the vero cells and ipec-j2 cells were pre-cooled at 4 °c for 15 min, incubated with pedv strains at 4 °c for 1 h, shifted to 37 °c to initiate internalization. the non-internalized viral particles were removed by washing. 30 min after shifting, the cells were fixed and stained with anti-pedv-s (red) and anti-eea1 (green) primary antibodies (c). 40 min after shifting, the cells were fixed and stained with anti-pedv-s (red) and anti-rab7 (green) primary antibodies (d). 50 min after shifting, the cells were fixed and stained with anti-pedv-s (red) and anti-lamp1 (green) primary antibodies (e). scale bars indicate 5 μm in c-e. *p < 0.05; **0.05 < p < 0.01; ***0.01 < p < 0.001; ****p < 0.001. 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 [23, 24] , 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 [32] . 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 [65] . 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-j2 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-j2 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 3.0) with proteinase k (1 mg/ml), and the latter exhibited a stronger capacity to remove viruses. the results of dynamic invasion showed that the virus invaded vero cells 100% within 60 min, while the invasion efficiency of the ipec-j2 cells was only approximately 30%. although ipec-j2 cells are considered the host cells of pedv, the cell lines cultured in vitro lost their polar growth state in vivo [66] , which possibly affects the viral recognition and reduces the infection efficiency of the virus. gds01 showed a lower invasion rate than gds09, 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 gds01 and gds09 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 gds01 and gds09 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 hepg2 cells and cos7 cells and phev entry into neuro-2a cells [23, 35] . 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-2 and eps 15. both cpz pre-treatment and sirna-mediated knockdown of chc and eps 15 can significantly reduce the invasion rate of pedv into cells, with no significant difference between the gds01 and gds09 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 15 were barely infected with pedv but cells overexpressing wild-type eps 15 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-1, gm1, 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 gds09 entry than gds01. the inhibitory effects of mβcd on vero cells were similar for gds09 and gds01 strains. mβcd effects were lower on gds09 than gds01 in ipec-j2 cells. we hypothesized that gds01 cell invasion mainly depended on cholesterol on the cell surface, while gds09 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-1, which plays a critical role in the process. dominant negative mutant, rna interference, and the cellular co-localization of caveolin-1 with viral particles provided further evidence that pedv entry needed caveolin-1. collectively, both subtypes of pedv entered the vero and ipec-j2 cells through caveolae-mediated endocytosis. however, park et al. [33] 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 20 μm [33] , while the highest concentration we used was 30 μm in vero cells. when the concentration is 10 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 [33] , 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-1 in the cells. as shown in figure 6b , pedv n protein is also present in the bottom layer when infected with ipec-j2 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 gds09 and gii subtype gds01 strains could enter vero and ipec-j2 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 gds01 and gds09 strains may be due to the difference of s gene especially the s1 region of s gene (homology was about 92%), 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 gds01 and gds09 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 4 cl and baf-a1 could inhibit pedv entry [33] , 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 eea1, rab7, and lamp1. liu et al. [67] 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-j2 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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springer nature remains neutral with regard to 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-j2 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-1. supplementary information accompanies this paper at https ://doi. org/10.1186/s1356 7-020-0739-7. and ipec-j2 cells were treated with different concentrations of dynasore at 37 °c for 4 h. cck-8 solution was added to each well at 37 °c for 1 h, and absorptions of 450 nm were detected. dmso was used as a negative control. (b) the vero cells and ipec-j2 cells were transfected with sidyn, and the second transfection was carried out at 24 h after the first transfection. the inference efficiency was detected by qrt-pcr and western blotting at 48 h after the first transfection. ctrl means control. **0.05 < p < 0.01; ***0.01 < p < 0.001; ****p < 0.001.additional file 2. clathrin-mediated endocytosis is involved in pedv entry. (a) vero cells and ipec-j2 cells were treated with different concentrations of cpz at 37 °c for 4 h. cck-8 solution was added to each well at 37 °c for 1 h, and absorptions of 450 nm were detected. double-distilled water was used as a negative control. (b, c) the vero cells and ipec-j2 cells were transfected with sichc and sieps15, and the second transfection was carried out at 24 h after the first transfection. the inference efficiency was detected by qrt-pcr and western blotting at 48 h after the first transfection. ctrl means control. *p < 0.05; **0.05 < p < 0.01; ***0.01 < p < 0.001; ****p < 0.001. cells were seeded in 12-well plates until confluence. cells were pre-treated with 30 μm cpz, 3 mm mβcd, 30 μm nystatin, 50 mm nh4cl and 200 nm baf a1 respectively, at 37 °c for 1 h and incubated with gds01 (a) and gds09 (b) strains for 1 h. double-distilled water was used as a negative control. the cells were collected at 9 hpi and detected by immunofluorescence staining against the pedv s protein (green). nuclei were stained with dapi (blue). scale bars indicate 100 μm. cells were seeded in 12-well plates until confluence. cells were pre-treated with 50 μm cpz, 1.5 mm mβcd, 50 μm nystatin, 50 mm nh4cl and 400 nm baf a1 respectively, at 37 °c for 1 h and incubated with gds01 (a) and gds09 (b) strains for 1 h. double-distilled water was used as a negative control. the cells were collected at 15 hpi and detected by immunofluorescence staining against the pedv s protein (green). nuclei were stained with dapi (blue). scale bars indicate 100 μm. the authors declare that they have no competing interests.received: 5 october 2019 accepted: 7 january 2020